TITLE MULTI-COMPONENT PERACID GENERATION SYSTEM

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application

Nos. 61/102,505; 61/102,512; 61/102,514; 61/102,520; 61/102,531 ; and 61/102,539; each filed October 3, 2008, each of which incorporated by reference herein in their entireties.

FiELD OF THE INVENTION

This invention relates to the field of enzymatic peroxycarboxylic acid synthesis and in situ enzyme catalysis. Specifically, a multi-component peroxycarboxylic acid generation system is provided for enzymatically producing aqueous solutions of peroxycarboxylic acids upon mixing the components from a first compartment with the components of a second compartment. At least one peroxycarboxylic acid is produced at sufficient concentrations as to be efficacious for the disinfection or sanitization of surfaces, medical instrument sterilization, food processing equipment sterilization, and suitable for use in laundry care applications such as bleaching, destaining, deodorizing, and sterilization.

BACKGROUND OF THE INVENTION

Peroxycarboxylic acid compositions have been reported to be effective antimicrobial agents. Methods to clean, disinfect, and/or sanitize hard surfaces, meat products, living plant tissues, and medical devices against undesirable microbial growth have been described (e.g., U.S. Patent 6,545,047; U.S. Patent 6,183,807; U.S. Patent 6,518,307; U.S. Patent 5,683,724; and U.S. Patent Application Publication No. 2003/0026846). Peroxycarboxylic acids have also been reported to be useful in preparing bleaching compositions for laundry detergent applications (e.g., U.S. Patent 3,974,082; U.S. Patent 5,296,161 ; and U.S. Patent 5,364,554). PeroxycarboxyJic acids can be prepared by the chemical reaction of a carboxylic acid and hydrogen peroxide (see Organic Peroxides, Daniel Swern, ed., Vol. 1 , pp 313-516; Wiley interscience, New York, 1971 ). The reaction is usually catalyzed by a strong inorganic acid, such as concentrated sulfuric acid. The reaction of hydrogen peroxide with a carboxylic acid is an equilibrium reaction, and the production of peroxycarboxylic acid is favored by the use of an excess concentration of peroxide and/or carboxyiic acid, or by the removal of water.

Some peroxycarboxylic acid-based disinfectants or bleaching agents are comprised of an equilibrium mixture of peroxycarboxylic acid, hydrogen peroxide, and the corresponding carboxylic acid. One disadvantage of these commercial peroxycarboxylic acid cleaning systems is that the peroxycarboxyϋc acid is oftentimes unstable in solution over time. One way to overcome the stability problem is to generate the peroxycarboxylic acid prior to use by combining multiple reaction components that are individually stable for extended periods of time. Preferably, the individual reaction components are easy to store, relatively safe to handle, and capable of quickly producing an efficacious concentration of peroxycarboxylic acid upon mixing.

One way to overcome the disadvantages of chemical peroxycarboxylic acid production is to use an enzyme catalyst having perhydrolysis activity. U.S. Patent Application No. 11/638,635 and U.S. Patent Application Publication Nos. 2008/0176783, 2008/0176299, and 2009/0005590 to DiCosimo et a/, disclose enzymes structurally classified as members of the CE- 7 family of carbohydrate esterases (Ae., cephalosporin C deacetylases [CAHs] and acetyl xylan esterases [AXEs]) that are characterized by significant perhydrolysis activity for converting carboxylic acid esters (in the presence of a suitable source of peroxygen, such as hydrogen peroxide) into peroxycarboxylic acids at concentrations sufficient for use as a disinfectant and/or a bleaching agent Some members of the CE-7 family of carbohydrate esterases have been demonstrated to have perhydrolytic activity sufficient to produce 4000 - 5000 ppm peracetic acid from acetyl esters of alcohols, diols, and glycerols in 1 minute and up to 9000 ppm between 5 minutes and 30 minutes once the reaction components were mixed (DiCosimo et a/., U.S. 2009/0005590).

Enzymatic peroxycarboxylic acid generation systems may be based on a two-component system, where each component is stored in a separate compartment until use. Typically, the enzyme catalyst having perhydrolysis activity is stored in one compartment with the carboxyiic acid ester substrate and the source of peroxygeπ (typically an aqueous solution of hydrogen peroxide) is stored in a second compartment. The components of the two compartments are mixed to produce the desired aqueous solution of peroxycarboxy I ic acid ,

However, mufti-component enzymatic peracid generation systems may also suffer from certain problems. One problem may be the use of one or more carboxyiic acid ester substrates that are insoluble or partially insoluble in water after mixing of the two components. The limited solubility of certain carboxyiic acid ester substrates can result in at least three conditions that interfere with the ability to efficaciously produce and deliver a peroxycarboxylic acid product: first, the viscosity of the enzyme catalyst/substrate constituent can be too high to permit efficient mixing with a second constituent comprising a source of peroxygen, which decreases the rate of production of peroxycarboxylic acid; second, the viscosity of the enzyme catalyst/substrate constituent can be too high to permit certain modes of delivery of a product comprising a mixture of the enzyme catalyst/substrate constituent and the source of peroxygen, such as spraying; third, the dissolution rate of the substrate in the enzyme/substrate component after mixing with a second component comprising a source of peroxygen in aqueous solution is too low to permit a satisfactory rate of production of peroxycarboxylic acid. The carboxyiic acid ester solubility problems also become evident in situations where use of a particular ratio of a component comprising an aqueous source of peroxygen to a component comprising an enzyme catalyst/substrate constituent is desired. As such, commercial uses of multi-component systems that involve the storage of the enzyme catalyst having perhydrolysis activity and substrate separately from a source of peroxygen until a desired time of reaction have remained impracticable for some applications. The use of organic cosolvents to enhance mixing and/or alter the viscosity of the carboxylic acid ester in water may be problematic. Organic solvents can be deleterious to the activity of enzymes, either when enzymes are suspended directly in organic solvents, or when miscible organic/aqueous single phase solvents are employed. Two literature publications that review the effects of organic solvents on enzyme activity and structure are: (a) C. Laane et al., Biotechnol. Bioeng. 30:81-87 (1987) and (b) Cowan, D.A. and Plant, A., Bbcatalysts in Organic Phase Systems., Ch. 7 in Biocatalvsis at Extreme Temperatures, Kelly, R.W.W, and Adams, M., eds., Amer. Chem. Soc. Symposium Series, Oxford University Press, New York, NY, pp 86-107 (1992). Cowan and Plant note (on page 87) that there is little or no value in using organic solvents having a log P < 2 to stabilize intracellular enzymes in an organic phase system. Organic solvents having a log P between two and four can be used on a case-by-case basis dependent on enzyme stability, and those having a log P > 4 are generally useful in organic phase systems.

Cowan and Plant, supra, further note (on page 91) that the effect of direct exposure of an enzyme dissolved in a single-phase organic-aqueous solvent depends on solvent concentration, solvent/enzyme surface group interactions, and solvent/enzyme hydration shell interactions. Because a solvent's log P value must be sufficiently low so that the solvent is fully miscible with the aqueous phase to produce a single-phase, a single-phase organic- aqueous solvent containing a low log P organic solvent usually has a negative effect on enzyme stability except in low organic solvent concentration applications. The storage stability of a CE-7 enzyme having perhydrolysis activity is a concern when stored in a carboxylic acid ester substrate or a mixture of the carboxylic acid ester and one or more cosolvents having a partition coefficient (as measured by a log P value, i.e., the logarithm of the partition coefficient of a substance between octanol and water, where P equals [soiute] _0Cta noi/[solute] _W ater) of two or less. Several of the organic ester substrates described by DiCostmo et al. in U.S. 2009/0005590 have log P values of less than two. For example, triacetin is reported to have a log P of 0.25 (Y. M. Gunning, et a!., J. Agric. Food Chem. 48:395-399 (2000)), similar to that of ethanol (log P -0.26) and isopropanol (log P 0.15) (Cowan and Plant); therefore the storage of enzyme powder in triacetiπ would be expected to result in unacceptable loss of enzyme activity, as would the use of additional cosolvents with log P < 2 (e.g., cyclohexanone, log P = 0.94) (Cowan and Plant); 1 ,2- propanediol, log P = -1.41 (Gunning, et al.); 1 ,3-propanediol, log P = -1.3 (S-J. Kuo, et al., J. Am. Oil Chem. Soc. 73: 1427-1433 (1996); diethylene glycol butyl ether, log P = 0.56 (N. Funasaki, et al., J. Phys. Chem. 88:5786-5790 (1984); triethyleneglycoi, log P = -1.75 (L. Braeken, et al., ChemPhysChem 6:1606- 1612 (2005)). Co-owned, co-filed, and copending U.S. Patent application under attorney docket number CL4205 US NA entitledΕNZYMATIC PERACID PRODUCTION USING A COSOLVENT" describes the use of organic cosolvents having a log P value of about 2 or less to control the viscosity of a substrate-containing component and to enhance the solubility of the substrate in an aqueous reaction mixture without causing substantial loss of perhydrolytic activity of the enzyme catalyst,

Co-owned, co-filed, and copending U.S. Patent applications under attorney docket numbers CL4386 US NA and CL4387 US NA, each having the title "STABILIZATION OF PERHYDROLASES", describe various ways to stabilize enzymatic perhydrolysis activity of enzyme powders when present in the carboxylic acid ester substrate component of a multi-component peroxycarboxylic acid generation system.

Co-owned, co-filed, and copending U.S. Patent application under attorney docket number CL4392 US NA entitled "IMPROVED PERHYDROLASES FOR ENZYMATIC PERACID GENERATION" describes variant CE-7 enzymes having improved perhydrolytic activity.

The problem to be solved is to provide multi-component peroxycarboxylic acid generation systems comprising a combination of ingredients characterized by enhanced storage stability of the CE-7 catalyst's perhydrolytic activity and/or improved mixing and/or viscosity characteristics of the carboxylic acid ester substrate-containing component. In a further embodiment, the muiti-component peroxycarboxylic acid generation system preferably comprises a variant CE-7 enzyme having improved perhydrolytic activity.

SUMMARY OF THE INVENTION The stated problem has been solved by providing a multi-component peroxycarboxylic acid generating system comprising a first compartment and a second compartment, wherein the components in the respective compartments provide enhanced storage stability and/or improved mixing characteristics. In one embodiment, a multi-component peroxycarboxyfic acid generation system is provided comprising: a first compartment comprising a first component and a second compartment comprising a second component and means for mixing the first component and the second component to produce an aqueous solution of peracetic acid; wherein the first component comprises (i) an enzyme powder comprising a formulation of:

(a) at least one enzyme catalyst having perhydrolysis activity, wherein said enzyme catalyst comprises an enzyme having a carbohydrate esterase family 7 (CE-7) signature motif that aligns with SEQ ID NO: 1 using CLUSTALW, said signature motif comprising:

(1) an RGQ motif at amino acid positions aligning with 1 18-120 of SEQ ID NO:1 ;

(2) a GXSQG motif at amino acid positions aligning with 179-183 of SEQ ID NO: 1 ; and (3) an HE motif at amino acid positions aligning with

298-299 of SEQ ID NO: 1 ; said enzyme comprising at least 30% amino acid identity to SEQ ID NO: 1; and

(b) at least one excipient; (ii) a carboxylic acid ester substrate selected from the group consisting of (a) one or more esters having the structure

wherein

X is an ester group of the formula ReC(O)O;

Re is a C1 to C7 linear, branched or cyclic hydrocarbyl moiety, optionally substituted with a hydroxy! group or C1 to C4 alkoxy group, wherein Re optionally comprises one or more ether linkages where Re is C2 to C7;

Rs is a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety optionally substituted with a hydroxyl group, wherein each carbon atom in R ₅ individually comprises no more than one hydroxyl group or no more than one ester group, and wherein R ₅ optionaliy comprises one or more ether linkages; m is 1 to the number of carbon atoms in R _5, said one or more esters having solubility in water of at least 5 ppm at 25 ⁰ C;

(b) one or more glycerides having the structure

0

-O -CH ₇ - -CH- -CH,- -OR,

OR ₃

wherein Ri is a C1 to C7 straight chain or branched chain alky! optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R ₃ and R ₄ are individually H or RiC(O);

(C) one or more esters of the formula

O

R ₁ - -O wherein Ri is a C1 to C7 straight chain or branched chain alkyi optionaily substituted with an hydroxyl or a C1 to C4 alkoxy group and R ₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, aikylaryi, alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n , or (CH ₂ CH(CH ₃ )-

O) _n H and n is 1 to 10;

(d) one or more acetylated monosaccharides, acetylated disaccharides, or acetylated polysaccharides; and

(e) any combination of (a) through (d); wherein the amount of the carboxylic acid ester substrate in the first component is designed to provide a final concentration of 0.5 wt % to 10 wt% in a reaction formulation formed by combining the first and second components;

(iii) a buffer selected from the group consisting of bicarbonate, citrate, acetate, phosphate, pyrophosphate, methylphosphonate, succinate, malate, fumarate, tartrate, and maleate; (iv) a cosolvent selected from the group consisting of tripropylene glycol methyi ether, dipropylene glycol methyl ether, propylene glycol methyl ether, diethylene glycol butyl ether, dipropylene glycol, triethylene glycol, 1 ,2-propanediol, N-ethyi-2-pyrroldinone, isopropanol, ethanot, ethyl lactate, 1 ,3-propanediol, and any combination thereof; and (v) optionally at least one surfactant; wherein the second component comprises water, hydrogen peroxide and a hydrogen peroxide stabilizer.

In another aspect, a multi-component peroxy carboxylic acid generation system is provided comprising a first compartment comprising a first component and a second compartment comprising a second component and means for mixing the first and second component to produce an aqueous solution of peracetic acid wherein the first component comprises (i) an enzyme powder comprising a formulation of

(a) at least one CE-7 enzyme having perhydrolysis activity, wherein said at least one CE-7 enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 19 and SEQ ID NO: 20 or an amino acid sequence substantially similar to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 19 or SEQ ID NO: 20; and

(b) at least one excipient;

(ii) a carboxylic acid ester substrate selected from the group consisting of monoacetin, diacetin, triacetin, and a mixtures thereof; wherein the amount of the carboxyiic acid ester substrate in the first component is designed to provide a finai concentration of 0.5 wt % to 10 wt% in a reaction formulation formed by combining the first and second components; (iii) a buffer selected from the group consisting of bicarbonate, citrate, acetate, phosphate, pyrophosphate, methylphosphonate, succinate, malate, fumarate, tartrate, and maleate;

(iv) a cosolvent selected from the group consisting of tripropySene glycol methyl ether, dipropylene glycol methyl ether, propylene glycol methyl ether, diethyiene glycol butyl ether, dipropylene glycol, Methylene glycol, 1 ,2-propanedioS, N-ethyl-2-pyrroidinone, isopropanol, ethanol, ethyl lactate, 1,3-propanedioi, and any combination thereof; and (v) optionally at least one surfactant; wherein the second component comprises water, hydrogen peroxide and a hydrogen peroxide stabilizer. In another aspect, the multi-component peroxycarboxylic acid generation system above is provided wherein:

(i) the at least one CE-7 enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 19 and SEQ ID NO: 20, wherein amino acid residue 277 of SEQ ID NO: 19 or SEQ !D NO: 20 is selected from the group consisting of alanine, valine, serine, and threonine; (ii) the carboxylic acid ester substrate is triacetin; wherein the amount of triacetin in the first component is designed to provide a final concentration of 0,5 wt % to 10 wt% in a reaction formulation formed by combining the first and second components; (iii) the buffer is in a concentration of about 0.1 wt% to about 10% wt of the first component and said buffer is selected from the group consisting of sodium bicarbonate, potassium bicarbonate, a mixture of sodium bicarbonate and potassium bicarbonate, sodium phosphate, potassium phosphate, and a mixture of sodium phosphate and potassium phosphate;

(iv) the cosoivent is tripropylene glycol methy! ether and is in a concentration of up to 80 wt% of the first component; (vi) the surfactant is present and is polysorbate 80; and (vi) the hydrogen peroxide in the second component is present in an amount that provides a final concentration in a reaction formulation formed by combining the first and second components of from 0.33 wt% to about 30 wt%. In another aspect, the multi-component peroxycarboxylic acid generation system further comprises means for applying the aqueous solution of peracetic acid produced by mixing the first and second components to a surface for bleaching or disinfection.

In addition, a method of using the multi-component peroxycarboxylic acid generation system is provided comprising

(a) employing the means for mixing the first and second components whereby an aqueous formulation comprising peracetic acid is produced; and

(b) applying the aqueous formulation comprising peracetic acid produced in (a) to a surface, an article of clothing or a textile for bleaching, stain removal, odor reduction, sanitization, disinfection, or a combination thereof. BRIEF DESCRiPTlON OF THE FIGURES Figures 1 a and 1b depict an exemplary system for producing peroxycarboxylic acid in accordance with the present invention using a two compartment spray bottle. Referring to Figure 1a, the generic spray bottle system comprises a first chamber [1] containing a first component [2] and a second chamber [3] containing a second component [4], Transfer tubing [5] is used to transport the first and second components to a flow control elements [6] and [7], preferably having a single adjustment control knob [8] that allows the two components to be mixed at a desired v/v or w/w ratio. Figure 1 a depicts a pump [9] that pumps and mixes both liquid components prior to exiting the spray nozzle [10]. Figure 1 b is similar to Figure 1a except that the pump [9] and nozzle [10] are configured to delay mixing of the first and second components at the spray nozzle or on the target surface.

Figures 2a and 2b depict an exemplary system for producing peroxycarboxylic acid using mufti-compartment packets. The barrier materials form the exterior barrier of the multi-compartment system [11] and/or the internal compartment barrier [12] separating the first component [2] and the second component [4] are designed to be easily degradable and/or breakable (mechanically, chemically, and/or thermally) to generate the aqueous peroxycarboxylic acid mixture. Referring to Figure 2a, the delivery system comprises a dissolvable package comprising a breakable exterior barrier [11] and an internal compartment having an internal compartment barrier [12] comprising a first component [2] that is surrounded by a second component [4]. The internal compartment barrier [12] is designed to be breakable and/or degradable to mix the two components to produce the desired peroxycarboxylic acid. Figure 2b illustrates a dissolvable packet having the two compartments separated in parallel. As depicted in Figure 2b, the 2 compartment packet comprises a dissolvable and/or breakable exterior barrier [11] and a first and second compartments [1] and [3] containing components [2], [4] respectively. The first and second compartments separated using a barrier [13].

Figures 3a and 3b depict an exemplary system for producing peroxycarboxylic acid according to the present invention using a multi- compartment, flexible squeeze packet/bottle. Referring to Figure 3a, a manually deformable container [16] comprises a non-rigid wall [14]. Within container [16] is a manually disruptable/breakable internal compartment [15] containing a first component, wherein compartment [15] is surrounded by a second component [4]. Disruption of the internal compartment [15] permits mixing and generation of the activate material. As shown in Figure 3a, the manually deformable container (e.g., a plastic squeeze bottle) further comprises a directional spout with a removable stopper [17]. Figure 3b depicts a non-rigid container [16] comprising a first compartment [1] comprising a first component [2] and a second compartment [3] comprising a second component [4] wherein the two compartments are separated in a parallel configuration. The manually deformable bottle in Figure 3b is also depicted to have a directional spout [18] and a removable stopper [17], wherein the two components are mixed outside of the bottle.

BRIEF DESCRIPTION QF THE BIOLOGICAL SEQUENCES The following sequences comply with 37 C. F. R, §§ 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures - the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the European Patent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5(a~bis), and Section 208 and Annex C of the Administrative Instructions. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C. F. R. § 1.822.

SEQ ID NO:1 is the deduced amino acid sequence of a cephalosporin C deacetylase from Bacillus subtilis ATCC ^® 31954™.

SEQ ID NO:2 is the deduced amino acid sequence of a cephalosporin C deacetylase from B. subtilis ATCC ^® 6633™. SEQ ID NO:3 is the deduced amino acid sequence of a cephalosporin C deacetylase from B. licheniformis ATCC ^® 14580™.

SEQ ID NO:4 is the deduced amino acid sequence of an acetyl xylan esterase from B. pumilus PS213. SEQ ID NO:5 is the deduced amino acid sequence of an acetyl xylan esterase from Clostridium thermocelium ATCC ^® 27405™.

SEQ ID NO:6 is the deduced amino acid sequence of an acetyl xylan esterase from Thermotoga neapolitana. SEQ ID NO:7 is the deduced amino acid sequence of an acetyl xylan esterase from Thermotoga maritima MSB8.

SEQ ID NO:8 is the deduced amino acid sequence of an acetyl xylan esterase from Thermoanaerobacteήum sp. JW/SL YS485.

SEQ ID NO:9 is the deduced amino acid sequence of a cephalosporin C deacetylase from Bacillus sp. NRRL B-1491 1. It should be noted that the nucleic acid sequence encoding the cephalosporin C deacetylase from Bacillus sp. NRRL B-14911 as reported in GEN BANK ^® Accession number ZP_01168674 appears to encode a 15 amino acid N-terminal addition that is likely incorrect based on sequence alignments with other cephalosporin C deacetylases and a comparison of the reported length (340 amino acids) versus the observed length of other CAH enzymes (typically 318-325 amino acids in length; see co-owed, co-filed, and copending U.S. Patent Application under attorney docket number CL4205 US NA entitled "ENZYMATIC PERACID PRODUCTION USING A COSOLVENT"; herein incorporated by reference) . As such, the deduced amino acid sequence reported herein for the cephalosporin C deacetylase sequence from Bacillus sp. NRRL B-14911 does not include the N-terminal 15 amino acids as reported under GENBANK ^® Accession number ZPJ)1168674.

SEQ ID NO: 10 is the deduced amino acid sequence of a cephalosporin C deacetylase from Bacillus halodurans C-125.

SEQ ID NO:11 is the deduced amino acid sequence of a cephalosporin C deacetylase from Bacillus clausii KSM-K16.

SEQ ID NO: 12 is the deduced amino acid sequence of a Bacillus subtilis ATCC ^® 29233™ cephalosporin C deacetylase (CAH). SEQ ID NO:13 is the deduced amino acid sequence of a

Thermoanearobacterium saccharolyticum cephalosporin C deacetylase.

SEQ ID NO: 14 is the deduced amino acid sequence of a Thermotoga lettingae acetyl xylan esterase. SEQ ID NO: 15 is the deduced amino acid sequence of a Thermotoga petrophila acetyl xylan esterase.

SEQ ID NO: 16 is the deduced amino acid sequence of a first acetyl xyfan esterase from Thermotoga sp. RQ2 described herein as "RQ2{a)'\ SEQ ID NO:17 is the deduced amino acid sequence of a second acetyl xylan esterase from Thermotoga sp. RQ2 described herein as "RQ2{b)".

SEQ ID NO:18 is the amino acid sequence of the region encompassing amino acids residues 1 18 through 299 of SEQ iD NO:1.

SEQ ID NO:19 is the deduced amino acid sequence of a Thermotoga neapolitana acetyl xylan esterase variant from co-owned, co-filed, and copending U.S. Patent Application Attorney Docket No. CL4392 US NA entitled "IMPROVED PERHYDROLASES FOR ENZYMATIC PERACID GENERATION" (incorporated herein by reference in its entirety), where the Xaa residue at position 277 is AIa ₁ VaI, Ser, or Thr. SEQ iD NO:20 is the deduced amino acid sequence of a Thermotoga maritime MSB8 acetyl xylan esterase variant from co-owned, co-filed, and copending U. S, Patent Application Attorney Docket No. CL4392 US NA, where the Xaa residue at position 277 is Ala, VaI, Ser, or Thr.

SEQ ID NO:21 is the deduced amino acid sequence of a Thermotoga lettingae acetyl xyfan esterase variant from co-owned, co-filed, and copending U.S. Patent Application Attorney Docket No. CL4392 US NA, where the Xaa residue at position 277 is Ala, VaI, Ser, or Thr.

SEQ ID NO:22 is the deduced amino acid sequence of a Thermotoga petrophila acetyl xyian esterase variant from co-owned, co-filed, and copending U.S. Patent Application Attorney Docket No. CL4392 US NA, where the Xaa residue at position 277 is AIa ₁ VaI, Ser, or Thr.

SEQ ID NO:23 is the deduced amino acid sequence of a Thermotoga sp. RQ2 acetyl xylan esterase variant derived from"RQ2(a)" from co-owned, co-filed, and copending U.S. Patent Application Attorney Docket No. CL4392 US NA, where the Xaa residue at position 277 is Ala, VaI, Ser, or Thr.

SEQ ID NO:24 is the deduced amino acid sequence of a Thermotoga sp. RQ2 acetyl xylan esterase variant derived from "RQ2{b)" from co-owned, co-fiied, and copending U.S. Patent Application Attorney Docket No. CL4392 US NA, where the Xaa residue at position 278 is Ala, VaI, Ser, or Thr.

SEQ ID NO:25 is the deduced amino acid sequence of an acetyl xylan esterase from Thermoanaerobacterium sp. JW/SL YS485. SEQ ID NO:26 is the coding region of a kanamycin resistance gene

(/can) from Streptomyces kanamyceticus,

SEQ ID NO:27 is piasmid pKDi 3, which contains the kanamycin resistance gene.

SEQ iD NO:28 is a forward primer used to done katG from piasmid pKD13.

SEQ iD NO;29 is a reverse primer used to clone katG from piasmid pKD13.

SEQ ID NO: 30 is the PCR product of the katG amplification from piasmid pKD13 using the primers of SEQ ID NO:28 and SEQ ID NO:29. SEQ iD NO:31 is the coding region of the catalase-peroxidase gene

{katG).

SEQ ID NO:32 is the deduced amino acid sequence of katG.

SEQ ID NO:33 is piasmid pKD46, which contains the λ-Red recombinase genes. SEQ ID NO:34 is a forward primer used to confirm disruption of katG.

SEQ ID NO:35 is a reverse primer used to confirm disruption of katG.

SEQ ID NO:36 is the temperature-sensitive piasmid pCP20, which contains the FLP recombinase.

SEQ ID NO:37 is a forward primer used to clone katE from piasmid pKD13.

SEQ ID NO:38 is a reverse primer used to clone katE from piasmid pKD13.

SEQ ID NO:39 is the PCR product of the katE amplification from piasmid pKD13 using the primers of SEQ ID NO:37 and SEQ ID NO:38. SEQ ID NO:40 is the coding region of the catalase HPII gene (katE).

SEQ ID NO:41 is the deduced amino acid sequence of katE.

SEQ ID NO:42 is a forward primer used to confirm disruption of katE.

SEQ ID NO:43 is a reverse primer used to confirm disruption of katE. SEQ !D NO:44 is a coding region of a gene encoding acetyl xylan esterase from Thermotoga neapolitana as reported in GENBANK ^® (accession No. AE000512).

SEQ ID NO:45 is a forward primer used to amplify the acetyi xylan esterase gene from Thermotoga neapolitana.

SEQ ID NO:46 is a reverse primer used to amplify the acetyl xylan esterase gene from Thermotoga neapolitana.

SEQ ID NO:47 is the PCR product of the acetyi xylan esterase amplification using the primers of SEQ ID NO:45 and SEQ ID NO:46. SEQ ID NO:48 is a gene encoding acetyl xylan esterase from

Thermotoga maritima as reported in GENBANK ^® (accession No. NP_227893.1).

SEQ ID NO:49 is a forward primer used to amplify the acetyl xylan esterase gene from Thermotoga maritima. SEQ ID NO:50 is a reverse primer used to amplify the acetyl xylan esterase gene from Thermotoga maritima.

SEQ ID NO:51 is the PCR product of the acetyl xylan esterase amplification using the primers of SEQ ID NO:49 and SEQ ID NO:50.

SEQ ID NOs: 52 and 53 are forward and reverse primers as described in Example 25.

SEQ NO: 54 is the nucleic acid sequence of the nucleic acid product amplified by SEQ ID NO: 52 and 53 that was used to prepare plasmid pSW207.

SEQ ID NOs: 55 and 56 are forward and reverse primers as described in Example 25.

SEQ NO: 57 is the codon optimized Thermotoga maritima coding sequence used to prepare plasmid pSW228.

SEQ ID NOs 58-65 are forward and reverse primers used to prepare the coding sequences of the Thermotoga maritima variants in Example 26, DETAILED DESCRIPTION OF THE INVENTION

In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise. As used herein, the articles "a", "an", and "the" preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore "a", "an" and "the" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

As used herein, the term "comprising" means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term "comprising" is intended to include embodiments encompassed by the terms "consisting essentially of and "consisting of. Similarly, the term "consisting essentially of is intended to include embodiments encompassed by the term "consisting of. As used herein, the term "about" modifying the quantity of an ingredient or reactant employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents to the quantities.

Where present, ail ranges are inclusive and combinable. For example, when a range of "1 to 5" is recited, the recited range should be construed as including ranges "1 to 4", "1 to 3", "1-2", "1-2 & 4-5", "1-3 & 5", and the like.

As used herein, the terms "substrate", "suitable substrate", and "carboxylic acid ester substrate" interchangeably refer specifically to:

(a) one or more esters having the structure

wherein X is an ester group of the formula R ₆ C(O)O;

R ₆ is a C1 to C7 linear, branched or cyclic hydrocarbyl moiety, optionally substituted with a hydroxyl group or C 1 to C4 alkoxy group, wherein R ₆ optionally comprises one or more ether linkages where R ₆ is C2 to C7;

Rs is a C1 to C6 ϋπear, branched, or cyclic hydrocarbyl moiety optionally substituted with a hydroxyl group, wherein each carbon atom in R ₅ individually comprises no more than one hydroxyl group or no more than one ester group, and wherein R ₅ optionally comprises one or more ether linkages; m is 1 to the number of carbon atoms in R _5, said one or more esters having solubility in water of at least 5 ppm at 25 ⁰ C; or

(b) one or more glycerides having the structure

wherein Ri is a C1 to C7 straight chain or branched chain alky! optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R3 and R ₄ are individually H or R ₁ C(O); or

(c) one or more esters of the formula

wherein Ri is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxy! or a C1 to C4 alkoxy group and R ₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkyny!, aryi, afkylaryl, alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n , or (CH ₂ CH(CHa)-O) _n H and n is 1 to 10; or

(d) one or more acetylated monosaccharides, acetylated disacchartdes, or acetylated polysaccharides; or

(e) any combination of (a) through (d). Examples of said carboxylic acid ester substrate may include monoacetin; triacetin; monopropionin; dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucose pentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylan fragments; β-D-ribofuranose-1 ,2,3,5~tetraacetate; tri-O- acetyl-D-ga!actal; tri-O-acetyi-glucal; propylene glycol diacetate; ethylene glycol diacetate; monoesters or diesters of 1,2-ethanedιol; 1 ,2-propanediol; 1 ,3-propanediol; 1 ,2-butanediol; 1 ,3-butanedio!; 2,3-butanediol; 1 ,4-butanediol; 1 ,2-pentanediol; 2,5-pentanediol; 1,6-pentanediol, 1 ,2-hexanediol; 2,5- hexanediol; 1,6-hexanedio!; or any combination thereof.

As used herein, the term "peracid" is synonymous with peroxyacid, peroxycarboxylic acid, peroxy acid, percarboxylic acid and peroxoic acid.

As used herein, the term "peracetic acid" is abbreviated as "PAA" and is synonymous with peroxyacetic acid, ethaneperoxoic acid and all other synonyms of CAS Registry Number 79-21-0.

As used herein, the term "monoacetin" is synonymous with glycerol monoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term "diacetin" is synonymous with glycerol diacetate; glycerin diacetate, glyceryl diacetate, and all other synonyms of CAS Registry Number 25395-31-7.

As used herein, the term "triacetin" is synonymous with glycerin triacetate; glycerol triacetate; glyceryl triacetate, 1,2,3-triacetoxypropane; 1 ,2,3- propanetriol triacetate and all other synonyms of CAS Registry Number 102- 76-1. As used herein, the term "monobutyrin" is synonymous with glycerol monobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.

As used herein, the term "dibutyrin" is synonymous with glycerol dibutyrate and glyceryl dibutyrate. As used herein, the term "tributyrin" is synonymous with giycerol tributyrate, 1 ,2,3-tributyrylgIycerol, and all other synonyms of CAS Registry Number 60-01 -5.

As used herein, the term "monopropioniπ" is synonymous with giycerol monopropionate, glycerin monopropionate, and glyceryl moπopropionate.

As used herein, the term "dipropionin" is synonymous with glycerol dipropionate and glyceryl dipropionate.

As used herein, the term "tripropioπin" is synonymous with glyceryl tripropionate, glycerol tripropionate, 1,2,3-tripropioπylglycerol, and all other synonyms of CAS Registry Number 139-45-7.

As used herein, the term "ethy! acetate" is synonymous with acetic ether, acetoxyethane, ethyl ethaπoate, acetic acid ethyl ester, ethanoic acid ethyl ester, ethyl acetic ester and all other synonyms of CAS Registry Number 141-78-6. As used herein, the term "ethyl lactate" is synonymous with lactic acid ethyl ester and all other synonyms of CAS Registry Number 97-64-3.

As used herein, the terms "acetylated sugar" and "acetylated saccharide" refer to mono-, di- and polysaccharides comprising at least one acetyl group. Examples include, but are not limited to, glucose pentaacetate; xylose tetraacetate; acetylated xylan; acetyiated xylan fragments; β-D- ribofuranose-1 ,2,3,5-tetraacetate; tri-O-acetyl-D-galactaS; and tri-O-acetyl- glucal.

As used herein, the terms "hydrocarbyl", "hydrocarbyl group", and "hydrocarbyl moiety" is meant a straight chain, branched or cyclic arrangement of carbon atoms connected by single, double, or triple carbon to carbon bonds and/or by ether linkages, and substituted accordingly with hydrogen atoms. Such hydrocarbyl groups may be aliphatic and/or aromatic. Examples of hydrocarbyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl, cyclobutyl, peπtyl, cyclopentyi, methylcyclopentyl, hexyl, cyclohexyl, benzyl, and phenyl. In a preferred embodiment, the hydrocarbyl moiety is a straight chain, branched or cyclic arrangement of carbon atoms connected by single carbon to carbon bonds and/or by ether linkages, and substituted accordingly with hydrogen atoms. As used herein, the terms "monoesters" and "diesters" of 1 ,2-ethanediol; 1 ,2-propanediol; 1 ,3-propanedio!; 1,2-butaπediol; 1,3-butaπediol; 2,3- butanediol; 1 ,4-butanediol; 1 ,2-pentanediol; 2,5-peπtanediol; 1 ,6-pentanedioi; 1 ,2-hexanediol; 2,5-hexanediol; 1,6-hexanediol; and mixtures thereof, refer to said compounds comprising at least one ester group of the formula RC(O)O, wherein R is a C1 to C7 linear hydrocarbyl moiety. In one embodiment, the carboxylic acid ester substrate is selected from the group consisting of propylene glycol diacetate (PGDA), ethylene glycol diacetate (EDGA), and mixtures thereof. As used herein, the term "propylene glycol diacetate" is synonymous with 1 ,2-diacetoxypropaπe, propylene diacetate, 1 ,2-propanedioi diacetate, and al! other synonyms of CAS Registry Number 623-84-7.

As used herein, the term "ethylene glycol diacetate" is synonymous with 1 ,2-diacetoxyethane, ethylene diacetate, glycol diacetate, and all other synonyms of CAS Registry Number 1 11 -55-7.

As used herein, the terms "suitable enzymatic reaction mixture", "components suitable for in situ generation of a peroxycarboxylic acid", "suitable reaction components", "suitable aqueous reaction mixture", and "reaction mixture" refer to the materials and water in which the reactants and enzyme catalyst come into contact. The components of the suitable aqueous reaction mixture are provided herein and those skilled in the art appreciate the range of component variations suitable for this process. In one embodiment, the suitable enzymatic reaction mixture produces peroxycarboxylic acid in situ upon combining the reaction components. As such, the reaction components may be provided as a multi-component system wherein the reaction components remains separated until use. The design of systems and means for separating and means for mixing the first and second components and means for applying the reaction mixture formed by mixing or combining the components are known in the art and generally will depend upon the physical form of the individual reaction components. For example, multiple active fluids (liquid-liquid) systems typically use multi-chamber dispenser bottles or two- phase systems (U.S. Patent Application Publication No. 2005/0139608; U.S. Patent 5,398,846; U.S. Patent 5,624,634; U.S. Patent 6,391,840; E P. Patent 0807156B1 ; U.S. Patent Application Pubϋcation No. 2005/0008526; and PCT Publication No. WO 00/61713) such as found in some bleaching applications wherein the desired bleaching agent is produced upon mixing the reactive fluids. Other forms of muiti-component systems used to generate peroxycarboxylic acid may include, but are not limited to, those designed for one or more solid components or combinations of solid-liquid components, such as powders (e.g., U.S. Patent 5, 1 16,575), multi-layered tablets (e.g., U.S. Patent 6,210,639), water dissolvable packets having multiple compartments (e.g., U.S. Patent 6,995,125) and solid agglomerates that react upon the addition of water (e.g., U.S. Patent 6,319,888).

In another embodiment, the carboxylic acid ester in the first component is selected from the group consisting of monoacetin, diacetiπ, triacetin, and combinations thereof. In another embodiment, the carboxylic acid ester in the first component is an acetylated saccharide. In another embodiment, the enzyme catalyst in the first component is a particulate solid. In another embodiment, the first reaction component is a solid tablet or powder.

As used herein, the term "perhydrolysis" is defined as the reaction of a selected substrate with peroxide to form a peroxycarboxylic acid. Typically, inorganic peroxide is reacted with the selected substrate in the presence of a catalyst to produce the peroxycarboxyfic acid. As used herein, the term

"chemical perhydrolysis" includes perhydrolysis reactions in which a substrate (a peroxycarboxylic acid precursor) is combined with a source of hydrogen peroxide wherein peroxycarboxylic acid is formed in the absence of an enzyme catalyst. As used herein, the term "perhydrolase activity" refers to the catalyst activity per unit mass (for example, milligram) of protein, dry celi weight, or immobilized catalyst weight.

As used herein, "one unit of enzyme activity" or "one unit of activity" or "U" is defined as the amount of perhydrolase activity required for the production of 1 μmol of peroxycarboxylic acid product per minute at a specified temperature.

As used herein, the terms "enzyme catalyst" and "perhydrolase catalyst" refer to a catalyst comprising an enzyme having perhydrolysis activity and may be in the form of a whole microbial celi, permeabilized microbial cel!(s), one or more celi components of a microbial celi extract, partially purified enzyme, or purified enzyme. The enzyme catalyst may also be chemically modified ( e.g., by pegylation or by reaction with cross-linking reagents). The perhydrolase catalyst may also be immobilized on a soluble or insoluble support using methods well-known to those skilled in the art; see for example, Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA; 1997. As described herein, all of the present enzymes having perhydrolysis activity are structurally members of the carbohydrate family esterase family 7 {CE-7 family) of enzymes (Coutinho, P.M., Henrissat, B. "Carbohydrate-active enzymes: an integrated database approach" in Recent Advances in Carbohydrate Bioengiπeerinq, HJ. Gilbert, G. Davies, B. Henrissat and B. Sveπssoπ eds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3-12.). The CE-7 family of enzymes has been demonstrated to be particularly effective for producing peroxycarboxylic acids from a variety of carboxylic acid ester substrates when combined with a source of peroxygen (WO2007/070609 and U.S. Patent Application Publication Nos. 2008/0176299, 2008/176783, and 2009/0005590 to DiCosimo et al.\ each herein incorporated by reference in their entirety). Members of the CE-7 family include cephalosporin C deacetylases

(CAHs; E.C. 3.1.1.41) and acetyl xylan esterases (AXEs; E.C. 3.1.1.72). Members of the CE-7 esterase family share a conserved signature motif (Vincent etal. _: J. MoI. Biol., 330:593-606 (2003)). Perhydrolases comprising the CE-7 signature motif and/or a substantially similar structure are suitable for use in the present invention. Means to identify substantially similar biological molecules are well known in the art (e.g., sequence alignment protocols, nucleic acid hybridizations and/or the presence of a conserved signature motif). In one aspect, the perhydrolase includes an enzyme comprising the CE-7 signature motif and at least 30%, preferably at least 33%, more preferably at least 40%, even more preferably at least 42%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at ieast 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to one of the sequences provided herein.

As used herein, the terms "cephalosporin C deacetylase" and "cephalosporin C acetyl hydrolase" refer to an enzyme (E. C. 3.1.1.41) that catalyzes the deacetylation of cephalosporins such as cephalosporin C and 7- aminocephalosporanic acid (Mitsushima et at., (1995) Appl. Env. Microbiol. 61 (6):2224-2229). As described herein, several cephalosporin C deacetylases are provided having significant perhydrolysis activity.

As used herein, "acetyl xylan esterases" refers to an enzyme (E.C. 3.1.1.72; AXEs) that catalyzes the deacetylation of acetylated xylans and other acetylated saccharides. As illustrated herein, several enzymes classified as acetyl xyian esterases are provided having significant perhydrolysis activity. As used herein, the term "Bacillus subtilis ATCC ^® 31954™" refers to a bacterial cell deposited to the American Type Culture Collection (ATCC) having international depository accession number ATCC ^® 31954™. Bacillus subtilis ATCC ^® 31954™ has been reported to have an ester hydrolase ("diacetinase") activity capable of hydrolyzing glycerol esters having 2 to 8 carbon acyl groups, especially diacetin (U.S. patent 4,444,886; herein incorporated by reference in its entirety). As described herein, an enzyme having significant perhydrolase activity has been isolated from B. subtilis ATCC ^® 31954™ and is provided as SEQ ID NO:1. The amino acid sequence of the isolated enzyme has 100% amino acid identity to the cephalosporin C deacetylase provided by GENBANK ^® Accession No. BAA01729.1.

As used herein, the term "Bacillus subtilis ATCC ^® 29233™" refers to a strain of Bacillus subtilis deposited to the American Type Culture Collection (ATCC) having international depository accession number ATCC ^® 29233™. As described herein, an enzyme having significant perhydrolase activity has been isolated and sequenced from S. subtilis ATCC ^® 29233™ and is provided as SEQ !D NO:Ϊ2. As used herein, the term "Clostridium thermocellum ATCC ^® 27405™" refers to a strain of Clostridium thermocellum deposited to the American Type Culture Collection (ATCC) having international depository accession number ATCC ^® 27405™. The amino acid sequence of the enzyme having perhydroiase activity from C. thermocellum ATCC ^® 27405™ is provided as SEQ ID NO:5.

As used herein, the term "Bacillus subtilis ATCC ^® 6633™" refers to a bacterial cell deposited to the American Type Culture Collection (ATCC) having international depository accession number ATCC ^® 6633™. Bacillus subtilis ATCC ^® 6633™ has been reported to have cephalosporin acetyl hydrolase activity (U.S. patent 6,465,233). The amino acid sequence of the enzyme having perhydroiase activity from B. subtilis ATCC ^® 6633™ is provided as SEQ ID NO:2. As used herein, the term "Bacillus licheniformis ATCC 14580™" refers to a bacterial cell deposited to the American Type Culture Collection (ATCC) having international depository accession number ATCC 14580™. Bacillus licheniformis ATCC 14580™ has been reported to have cephalosporin acetyihydrolase activity. The amino acid sequence of the enzyme having perhydroiase activity from B. licheniformis ATCC 14580™ is provided as SEQ ID NO:3.

As used herein, the term "Bacillus pumilus PS213" refers to a bacterial cell reported to have acetyl xylan esterase activity (GENBANK ^® AJ249957). The amino acid sequence of the enzyme having perhydroiase activity from Bacillus pumilus PS213 is provided as SEQ ID NO:4.

As used herein, the term "Thermotoga neapolitana " refers to a strain of Thermotoga neapolitana reported to have acetyl xylan esterase activity (GENBANK ^® AAB70869). The amino acid sequence of the enzyme having perhydroiase activity from Thermotoga neapolitana is provided as SEQ ID NO: 6.

As used herein, the term "Thermotoga maritima MSB8" refers to a bacterial ceil reported to have acetyl xylan esterase activity (GENBANK ^® NP_227893.1). The amino acid sequence of the enzyme having perhydroiase activity from Thermotoga maritima MSB8 is provided as SEQ ID NO: 7. As used herein, the term "Bacillus clausii KSM-K16" refers to a bacterial cell reported to have cephalosporin-C deacetylase activity (GENBANK ^® YP_175265). The amino acid sequence of the enzyme having perhydrojase activity from Bacillus clausii KSM-K16 is provided as SEQ ID NO: 11. As used herein, the term "Thermoanearobacterium saccharolyticum" refers to a bacterial strain reported to have acetyl xylan esterase activity (GENBANK ^® S41858). The amino acid sequence of the enzyme having perhydrolase activity from Thermoanearobacterium saccharolyticum is provided as SEQ (D NO: 13.

As used herein, the term "Thermotoga lettingae" refers to a bacterial cell reported to have acetyl xylan esterase activity (GENBANK ^® CP000812). The deduced amino acid sequence of the enzyme having perhydrolase activity from Thermotoga lettingae is provided as SEQ ID NO: 14. As used herein, the term "Thermotoga petrophila" refers to a bacterial cell reported to have acetyl xylan esterase activity (GENBANK ^® CP000702). The deduced amino acid sequence of the enzyme having perhydrolase activity from Thermotoga petrophila is provided as SEQ ID NO: 15.

As used herein, the term "Thermotoga sp. RQ2" refers to a bacterial ceil reported to have acetyl xylan esterase activity (GENBANK ^® CP000969). Two different acetyl xylan esterases have been identified from Thermotoga sp. RQ2 and are referred to herein as "RQ2(a)" (the deduced amino acid sequence provided as SEQ ID NO: 16) and "RQ2(b)" (the deduced amino acid sequence provided as SEQ ID NO: 17). As used herein, an "isolated nucleic acid molecule", "isolated polynucleotide", and "isolated nucleic acid fragment" will be used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term "amino acid" refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify specific amino acids: Three-Letter One-Letter

Amino Acid Abbreviation Abbreviation

Alanine Ala A

Arginine Arg R

Asparagine Asπ N

Aspartic acid Asp D

Cysteine Cys C

Glutamine GIn Q

Glutamic acid GIu E

Glycine Giy G

Histidine His H

Isoleuciπe EIe I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine VaI V

Any amino acid or as defined herein Xaa X

As used herein, "substantially similar" refers to nucleic acid molecules wherein changes in one or more nucleotide bases results in the addition, substitution, or deletion of one or more amino acids, but does not affect the functional properties (i.e., perhydrolytic activity) of the protein encoded by the DNA sequence. As used herein, "substantially similar" also refers to an enzyme having an amino acid sequence that is at least 30%, preferably at least 33%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, yet even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences reported herein wherein the resulting enzyme retains the present functional properties {i.e., perhydroiytic activity). "Substantially similar" may also refer to an enzyme having perhydroiytic activity encoded by nucleic acid molecules that hybridize under stringent conditions to the nucleic acid molecules reported herein. It is therefore understood that the invention encompasses more than the specific exemplary sequences.

For example, it is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded protein are common. For the purposes of the present invention substitutions are defined as exchanges within one of the following five groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr

(Pro, GIy); 2. Polar, negatively charged residues and their amides: Asp, Asn, Giu,

GIn;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, lie, VaI (Cys); and

5. Large aromatic residues: Phe, Tyr, and Trp. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes which result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid) or one positively charged residue for another (such as lysine for arginine) can also be expected to produce a functionally equivalent product. In many cases, nucleotide changes which result in alteration of the N-terminai and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Moreover, the skilled artisan recognizes that substantially similar sequences are encompassed by the present invention, In one embodiment, substantially similar sequences are defined by their ability to hybridize, under stringent conditions (0.1 X SSC, 0.1% SDS, 65°C and washed with 2X SSC, 0.1% SDS followed by 0.1 X SSC, 0.1 % SDS, 65°C) with the sequences exemplified herein. As used herein, a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single strand of the first molecule can anneal to the other molecule under appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J. and Russell, D., T. Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Stringency conditions can be adjusted to screen for moderately similar molecules, such as homologous sequences from distantly related organisms, to highly similar molecules, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes typically determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50 ⁰ C for 30 min. A more preferred set of conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60 ⁰ C. Another preferred set of stringent hybridization. conditions is 0.1X SSC, 0.1 % SDS, 65°C and washed with 2X SSC, 0.1% SDS followed by a final wash of 0.1X SSC, 0.1% SDS, 65°C with the sequences exemplified herein.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (Sambrook and Russell, supra). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (Sambrook and Russell, supra). In one aspect, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably, a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides in length, more preferably at least about 20 nucleotides in length, even more preferably at least 30 nucleotides in length, even more preferably at least 300 nucleotides in length, and most preferably at least 800 nucleotides in length. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

As used herein, the term "percent identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to, those described in: Computational Molecular Biology (Lesk, A, M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M, and Devereux, J., eds.) Stockton Press, NY (1991). Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megaϋgn program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl), the AlignX program of Vector NTI v. 7.0 (Informax, Inc., Bethesda, MD), or the EMBOSS Open Software Suite (EMBL-EBI; Rice et al., Trends in Genetics 16, (6):276-277 (2000)). Multiple alignment of the sequences can be performed using the CLUSTAL method (such as CLUSTALW; for example version 1.83) of alignment (Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res. 22:4673-4680 (1994); and Chenna et ai, Nucleic Acids Res 31 (13):3497-500 (2003)), available from the European Molecular Biology Laboratory via the European Bioinformatics Institute) with the default parameters. Suitable parameters for CLUSTALW protein alignments include GAP Existence penalty=15, GAP extension =0.2, matrix = Gonπet (e.g., Gonnet250), protein ENDGAP = -1 , Protein GAPDIST=4, and KTUPLE=L In one embodiment, a fast or slow alignment is used with the default settings where a slow alignment is preferred. Alternatively, the parameters using the CLUSTALW method (version 1.83) may be modified to also use KTUPLE =1, GAP PENALTY=I O, GAP extension =1 , matrix - BLOSUM (e.g., BLOSUM64), WINDOW=S, and TOP DIAGONALS SAVED=S. in one aspect, suitable isolated nucleic acid molecules encode a polypeptide having an amino acid sequence that is at least about 30%, preferably at least 33%, preferably at least 40%, preferably at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at (east 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences reported herein. Suitable nucleic acid molecules not only have the above homologies, but also typically encode a polypeptide having about 300 to about 340 amino acids, more preferably about 310 to about 330 amino acids, and most preferably about 318 to about 325 amino acids.

As used herein, the terms "signature motif, "CE-7 signature motif, and "diagnostic motif refer to conserved structures shared among a family of enzymes having a defined activity. The signature motif can be used to define and/or identify the family of structurally related enzymes having similar enzymatic activity for a defined family of substrates. The signature motif can be a single contiguous amino acid sequence or a collection of discontiguous, conserved motifs that together form the signature motif. Typically, the conserved motif(s) is represented by an amino acid sequence. As described herein, the present enzymes having perhydrolysis activity

("perhydrolases") belong to the family of CE-7 carbohydrate esterases

(DiCosimo et a/., U.S. Patent Application Publication No. 2009/0005590). As used herein, the phrase "enzyme is structurally classified as a CE-7 enzyme", "CE-7 perhydrolase" or "structurally classified as a carbohydrate esterase family 7 enzyme" will be used to refer to enzymes having perhydrolysis activity which are structurally classified as a CE-7 carbohydrate esterase. This family of enzymes can be defined by the presence of a signature motif (Vincent et a!., supra). The signature motif for CE-7 esterases comprises three conserved motifs (residue position numbering relative to reference sequence SEQ ID NO:

1): a) Arg1 18-Gly1 19-Gln120; b) Gly179-Xaa180-Ser181-Gln182-Gly183; and c) His298-Glu299. Typically, the Xaa at amino acid residue position 180 is glycine, alanine, proline, tryptophan, or threonine. Two of the three amino acid residues belonging to the catalytic triad are in bold, in one embodiment, the Xaa at amino acid residue position 180 is selected from the group consisting of glycine, alanine, proline, tryptophan, and threonine. Further analysis of the conserved motifs within the CE-7 carbohydrate esterase family indicates the presence of an additional conserved motif (LXD at amino acid positions 267-269 of SEQ ID NO:1) that may be used to further to define a perhydrolase belonging to the CE-7 carbohydrate esterase family. In a further embodiment, the signature motif defined above includes a fourth conserved motif defined as:

Leu267-Xaa268-Asp269.

The Xaa at amino acid residue position 268 is typically isoleucine, valine, or methionine. The fourth motif includes the aspartic acid residue (bold) belonging to the catalytic triad (Ser181-Asp269-His298). A number of well-known global alignment algorithms {i.e., sequence analysis software) may be used to align two or more amino acid sequences representing enzymes having perhydrolase activity to determine if the enzyme is comprised of the present signature motif. The aligned sequence(s) are compared to the reference sequence (SEQ ID NO:1) to determine the existence of the signature motif. In one embodiment, a CLUSTAL alignment (such as CLUSTALW) using a reference amino acid sequence (as used herein the perhydrolase sequence (SEQ ID NO:1) from the Bacillus subtilis ATCC ^® 31954™) is used to identify perhydrolases belonging to the CE-7 esterase family. The relative numbering of the conserved amino acid residues is based on the residue numbering of the reference amino acid sequence to account for small insertions or deletions (for example, five amino acids of iess) within the aligned sequence. Examples of other suitable algorithms that may be used to identify sequences comprising the present signature motif (when compared to the reference sequence) include, but are not limited to, Needleman and Wunsch (J. MoI. Biol. 48, 443-453 (1970); a global alignment tool) and Smith-Waterman {J. MoI. Biol, 147:195-197 (1981); a local alignment tool), in one embodiment, a Smith-Waterman alignment is implemented using default parameters. An example of suitable default parameters include the use of a BLOSUM62 scoring matrix with GAP open penalty = 10 and a GAP extension penalty = 0.5.

A comparison of the overall percent identity among perhydrolases exemplified herein indicates that enzymes having as little as 33% identity to SEQ ID NO: 1 (while retaining the signature motif) exhibit significant perhydrolase activity and are structurally classified as CE-7 carbohydrate esterases. In one embodiment, suitable perhydroiases include enzymes comprising the CE-7 signature motif and at least 30%, preferably at least 33%, more preferably at least 40%, even more preferably at least 42%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% _s 98%, or 99% amino acid identity to SEQ ID NO: 1.

Alternatively, a contiguous amino acid sequence comprising the region encompassing the conserved motifs may also be used to identify CE-7 family members.

As used herein, "codon degeneracy" refers to the nature of the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the present invention relates to any nucleic acid molecule that encodes all or a substantial portion of the amino acid sequences encoding the present microbial polypeptide. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

As used herein, the term "codon optimized", as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide for which the DNA codes.

As used herein, "synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are iigated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene. "Chemically synthesized", as pertaining to a DNA sequence, means that the component nucleotides were assembled in vitro, Manual chemical synthesis of DNA may be accomplished using well- established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequences to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

As used herein, "gene" refers to a nucleic acid molecule that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

As used herein, "coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3 ¹ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing site, effector binding site and stem-loop structure. As used herein, "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. As used herein, the "3' non-coding sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences (normally limited to eukaryotes) and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of poiyadeπylic acid tracts (normally limited to eukaryotes) to the 3' end of the mRNA precursor,

As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid molecule so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence, i.e., the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecule of the invention. Expression may also refer to translation of mRNA into a polypeptide.

As used herein, "transformation" refers to the transfer of a nucleic acid molecule into the genome of a host organism, resulting in genetically stable inheritance, In the present invention, the host cell's genome includes chromosomal and extrachromosomal (e.g., plasmid) genes. Host organisms containing the transformed nucleic acid molecules are referred to as "transgenic" or "recombinant" or "transformed" organisms. As used herein, the terms "plasmid", "vector" and "cassette" refer to an extrachromosomal element often carrying genes which are not part of the central metabolism of the ce!l, and usually in the form of circular double- stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA ₁ derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

As used herein, the term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to, the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wl), BLASTP ₁ BLASTN, BLASTX (Aitschul et al., J. MoI. Biol, 215:403-410 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wl 53715 USA), CLUSTALW (for example, version 1.83; Thompson et a/., Nucleic Acids Research, 22(22):4673-4680 (1994), and the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111 -20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY), Vector NTI (Informax, Bethesda, MD) and Sequencher v. 4.05. Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters set by the software manufacturer that originally load with the software when first initialized.

As used herein, the term "biological contaminants" refers to one or more unwanted and/or pathogenic biological entities including, but not limited to, microorganisms, spores, viruses, prions, and mixtures thereof. The process produces an efficacious concentration of at least one percarboxylic acid useful to reduce and/or eliminate the presence of the viable biological contaminants. In a preferred embodiment, the biological contaminant is a viable pathogenic microorganism. As used herein, the term "disinfect" refers to the process of destruction of or prevention of the growth of biological contaminants. As used herein, the term "disinfectant" refers to an agent that disinfects by destroying, neutralizing, or inhibiting the growth of biological contaminants. Typically, disinfectants are used to treat inanimate objects or surfaces. As used herein, the term

"disinfection" refers to the act or process of disinfecting, As used herein, the term "antiseptic" refers to a chemical agent that inhibits the growth of disease- carrying microorganisms. In one aspect, the biological contaminants are pathogenic microorganisms. As used herein, the term "sanitary" means of or relating to the restoration or preservation of health, typically by removing, preventing or controlling an agent that may be injurious to health. As used herein, the term "sanitize" means to make sanitary. As used herein, the term "sanitizer" refers to a sanitizing agent. As used herein the term "sanitization" refers to the act or process of sanitizing.

As used herein, the term "virucide" refers to an agent that inhibits or destroys viruses, and is synonymous with "viricide". An agent that exhibits the ability to inhibit or destroy viruses is described as having "virucidal" activity. Peroxycarboxylic acids can have virucidal activity. Typical alternative virucides known in the art which may be suitable for use with the present invention include, for example, alcohols, ethers, chloroform, formaldehyde, phenols, beta propiolactoπe, iodine, chlorine, mercury salts, hydroxylamiπe, ethylene oxide, ethylene glycol, quaternary ammonium compounds, enzymes, and detergents. As used herein, the term "biocide" refers to a chemical agent, typically broad spectrum, which inactivates or destroys microorganisms. A chemical agent that exhibits the ability to inactivate or destroy microorganisms is described as having "biocidal" activity. Peroxycarboxylic acids can have biocidal activity. Typical alternative biocides known in the art, which may be suitable for use in the present invention include, for example, chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone, acrolein, amines, chlorinated pheπolics, copper salts, orgaπo-sulphur compounds, and quaternary ammonium salts. As used herein, the phrase "minimum biocidal concentration" refers to the minimum concentration of a biocidal agent that, for a specific contact time, wiil produce a desired lethal, irreversible reduction in the viable population of the targeted microorganisms. The effectiveness can be measured by the !og ₁₀ reduction in viable microorganisms after treatment. In one aspect, the targeted reduction in viable microorganisms after treatment is at least a 3-iog reduction, more preferably at least a 4-log reduction, and most preferably at least a 5-log reduction. In another aspect, the minimum biocidal concentration is at least a 6-log reduction in viable microbial cells. As used herein, the terms "peroxygen source" and "source of peroxygen" refer to compounds capable of providing hydrogen peroxide at a concentration of about 1 mM or more when in an aqueous solution including, but not limited to, hydrogen peroxide, hydrogen peroxide adducts (e.g., urea- hydrogen peroxide adduct (carbamide peroxide)), perborates, and percarbonates. As described herein, the concentration of the hydrogen peroxide provided by the peroxygen compound in the aqueous reaction formulation is initially at least 1 mM or more upon combining the reaction components. In one embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 10 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 100 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 200 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is 500 mM or more. In yet another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is 1000 mM or more. The molar ratio of the hydrogen peroxide to enzyme substrate, e.g., triglyceride, (H ₂ O ₂ :substrate) in the aqueous reaction formulation may be from about 0.002 to 20, preferably about 0.1 to 10, and most preferably about 0.5 to 5.

By "oligosaccharide" is meant compounds containing between 2 and at least 24 monosaccharide units linked by glycosidic linkages. The term

"monosaccharide" refers to a compound of empirical formula (CH ₂ O) _n , where n>3, the carbon skeleton is unbranched, each carbon atom except one contains a hydroxyl group, and the remaining carbon atom is an aldehyde or ketone at carbon atom 2. The term "monosaccharide" also refers to intracellular cyclic hemiacetal or hemiketal forms.

As used herein, the term "excipient" refers to an inactive substance used to stabilize the active ingredient in a formulation, such as the storage stability of the active ingredient Excipients are also sometimes used to bulk up formulations that contain active ingredients. As described herein, the "active ingredient" is an enzyme catalyst comprising at least one enzyme having perhydrolysis activity, in one embodiment, the active ingredient is at least one CE-7 carbohydrate esterase having perhydrolysis activity. As used herein, the term "oligosaccharide excipieπt" means an oligosaccharide that, when added to an aqueous enzyme solution, improves recovery/retention of active enzyme (i.e., perhydrolase activity) after spray drying and/or improves storage stability of the resulting spray-dried enzyme powder or a formulation of the enzyme powder and an organic substrate. In one embodiment, the addition of the oligosaccharide excipient prior to spray drying improves the storage stability of the enzyme when stored in the carboxylic acid ester (i.e., a storage mixture substantially free of water). The carboxylic acid ester may contain a very low concentration of water, for example, triacetin typically has between 180 ppm and 300 ppm of water. As used herein, the phrase "substantially free of water" will refer to a concentration of water in a formulation of the enzyme powder and the carboxyiic acid ester that does not adversely impact the storage stability of enzyme powder when present in the carboxylic acid ester. In a further embodiment, "substantially free of water" may mean less than 2000 ppm, preferably less than 1000 ppm, more preferably less than 500 ppm, and even more preferably less than 250 ppm of water in the formulation comprising the enzyme powder and the carboxylic acid ester. Multi-component Peroxycarboxylic acid Generation Systems

Peroxycarboxylic acids are quite reactive and generally decrease in concentration over time. This is especially true for commercial pre-formed peroxycarboxylic acid compositions that often lack long term stability. Aqueous solutions of pre-formed peroxycarboxylic acids may also present handling and/or shipping difficulties, especially when shipping large containers and/or highly concentrated peroxycarboxyϋc acid solutions over longer distances. Further, pre-formed peroxycarboxyiic acid solutions may not be able to provide the desired concentration of peroxycarboxyiic acid for a particular target application. As such, it is highly desirable to keep the various reaction components separated, especially for liquid formulations.

The use of multi-component peroxycarboxyiic acid generation systems comprising two or more components that are combined to produce the desired peroxycarboxyiic acid has been reported. The individual components should be safe to handle, easy to ship, and stable for extended periods of time (i.e., as measured by the concentration of peroxycarboxyiic acid produced upon mixing). In one embodiment, the storage stability of a multi-component enzymatic peroxycarboxyiic acid generation system may be measured in terms of enzyme catalyst stability.

The design of systems and means for separating and combining multiple active components are known in the art and generally will depend upon the physical form of the individual reaction components. For example, multiple active fluids (liquid-liquid) systems typically use πiuiti-ch amber dispenser bottles or two-phase systems (e.g., U.S. Patent Application Pub. No. 2005/0139608; U.S. Patent 5,398,846; U.S. Patent 5,624,634; U.S. Patent 6,391 ,840; E.P. Patent 0807156B1 ; U.S. Patent Application. Pub. No.

2005/0008526; and PCT Publication No. WO 00/61713A1) such as found in some bleaching applications wherein the desired bleaching agent is produced upon mixing the reactive fluids. Other forms of mufti-component systems used to generate peroxycarboxyiic acid may include, but are not limited to, those designed for one or more solid components or combinations of solid-liquid components, such as powders (e.g., U.S. Patent 5,116,575), multi-layered tablets (e.g., U.S. Patent 6,210,639), water dissolvable packets having multiple compartments (e.g., U.S. Patent 6,995,125) and solid agglomerates that react upon the addition of water (e.g., U.S. Patent 6,319,888). United States Published Patent Application No. 2004-0127381 to

Scialia et a/, describes an aqueous two component laundry product. One part of the two part laundry product comprises a liquid cleaning composition while the second part comprises a bleaching composition; wherein the bleaching composition comprises an equiiibrium peroxycarboxylic acid solution comprising a carboxyϋc acid, a source of peroxygen, and the corresponding peroxycarboxylic acid.

A multi-component peroxycarboxylic acid generation system is provided herein that uses an enzyme catalyst to rapidly produce an aqueous peroxycarboxylic acid solution having a desired peroxycarboxylic acid concentration. The mixing may occur immediately prior to use and/or at the site (in situ) of application. The system wil) be comprised of at least two components that remain separated until use. Mixing of the components rapidly forms an aqueous peroxycarboxylic acid solution. The composition of each component is designed so that the resulting aqueous peroxycarboxylic acid solution comprises an efficacious peroxycarboxylic acid concentration suitable for the intended end use (e.g., disinfecting, stain removal, odor reduction, and bleaching applications). The composition of the individual components should be designed to (1) provide extended storage stability and (2) provide the ability to enhance formation of a suitable aqueous reaction formulation comprised of peroxycarboxyfic acid.

In one embodiment, the multi-component peroxycarboxylic acid generation system comprises a two component generation system. Various two component generation systems have been reported. In another embodiment, the two component generation system is substantially a two liquid component generation system. At a minimum, the present multi-component peroxycarboxylic acid generation system comprises (1) at least one enzyme catalyst having perhydrolysis activity, wherein said at least one enzyme is structurally classified as a CE-7 esterase, (2) a carboxyiic acid ester substrate, and (3) an aqueous source of peroxygen wherein the generation system enzymatically produces the desired peroxycarboxylic acid upon combining the components.

The ingredients and concentration of the ingredients within two component system should to be carefully selected and balanced to provide (1) storage stability of each component, especially the perhydrolysis activity of the enzyme catalyst and (2) physical characteristics that enhance solubility and/or the ability to effectively form the desired aqueous peroxycarboxylic acid solution {e.g., ingredients that enhance the solubility of the ester substrate in the aqueous reaction formulation and/or ingredients that modify the viscosity and/concentration of at least one of the liquid components [i.e. at least one cosolvent that does not have a significant, adverse effect on the enzymatic perhydrolysis activity]).

A multi-component peroxycarboxylic acid generation system is provided herein. The multi-component generation system may be comprised of at least two substantially liquid components. In one embodiment, the muiti-component generation system is a two component system comprising a first liquid component and a second liquid component. The use of the terms "first" or "second" liquid component is relative provided that two different liquid components comprising the specified ingredients remain separated until use.

Co-owned, co-filed, and copending U.S. Patent Application Attorney Docket No. CL4205 US NA entitled "ENZYMATIC PERAClD PRODUCTION USiNG A COSOLVENT" describes the use of at least one cosolvent to enhance solubility and/or the mixing characteristics of the ester substrate. The component comprising the carboxylic acid ester substrate and the perhydrolase catalyst comprises an organic solvent having a Log P value of less than about 2, wherein Log P is defined as the logarithm of the partition coefficient of a substance between octanol and water, expressed as P =

[solute]octaπof/[solute]waier- Several cosolvents having a log P value of 2 or less that do not have a significant adverse impact on enzyme activity are described. In one embodiment, the cosolvent is about 20 wt% to about 70 wt% within the reaction component comprising the carboxylic acid ester substrate and the enzyme.

Co-owned, co-filed, and copending U.S. Patent Application Attorney Docket No. CL4386 US NA entitled "STABILIZATION OF PERHYDROLASES" describes the use of a two component system wherein the first component comprises a formulation of a liquid carboxylic acid ester and solid enzyme powder comprising a formulation of (a) at least one CE-7 esterase having perhydrolysis activity and (b) at least one oligosaccharide excipient; and the second component comprises water having a source of peroxygen and a hydrogen peroxide stabilizer. In one embodiment, the at least one oligosaccharide has a number average molecular weight of at least about 1250 and a weight average molecuiar weight of at least about 9000. In another embodiment, the oligosaccharide excipient has a number average molecular weight of at least about 1700 and a weight average molecular weight of at least about 15000. In another embodiment, the oligosaccharide is maltodextrin. Co-owned, co-fiied, and copending U.S. Patent Application Attorney Docket No. CL4387 US NA also entitled "STABILIZATION OF PERHYDROLASES" describes the use of a two component system wherein the first component comprises a formulation of a liquid carboxyϋc acid ester and solid enzyme powder comprising a formulation of (a) an enzyme powder comprising at least one CE-7 esterase having perhydrolysis activity and at least one excipient and optionally at least one surfactant; and (b) at least one buffer, where in a preferred embodiment the buffer is added as a separate (i.e. separate from the enzyme powder) insoluble component to the carboxylic acid ester substrate; and the second component comprises water having a source of peroxygen and a hydrogen peroxide stabilizer. In one embodiment, the at least one excipient is an oligosaccharide that has a number average molecuiar weight of at least about 1250 and a weight average molecular weight of at ieast about 9000. In another embodiment, the at least one excipient is an oligosaccharide that has a number average molecular weight of at least about 1700 and a weight average molecular weight of at least about 15000. In another embodiment, the oligosaccharide is maltodextrin. In a further embodiment, the pH buffer is a bicarbonate buffer, In yet a further embodiment, the hydrogen peroxide stabilizer is TURPINAL ^® SL. Compositions and methods of using enzymes having perhydrolysis activity that are structurally classified as being members of the CE-7 carbohydrate esterase family have reported. These "perhydrolase" enzymes are particularly effective in producing peroxycarboxylic acids from a variety of ester substrates when combined with a source of peroxygen (See co-pending and co-owned published International Patent Application no. WO2007/070609 and U.S. Patent Application Publication Nos. 2008/0176299, 2008/176783, and 2009/0005590 to DiCosimo et a/.; each herein incorporated by reference in their entirety). The perhydrolases described in the published patent applications to DiCosimo et al. were al! based on amino acid sequences reported from previously reported CE-7 esterases (this group includes acetyl xyian esterases (AXEs) and cephalosporin acetyl hydrolases (CAHs)). Co- owned, co-filed, and copending U.S. Patent Application Attorney Docket No. CL4392 US NA discloses variants of several Thermotoga sp. CE-7 esterases having improved perhydrolysis activity and/or improved perhydrolysis activity to hydrolysis activity ratios (i.e., P/H ratios). In one embodiment, the two variant CE-7 perhydrolases have an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20.

Enzyme _, Powder

In one embodiment, the multi-component generation system comprises an enzyme powder comprising a formulation of at ieast one enzyme structurally classified as a CE-7 enzyme and having perhydrolysis activity and at least one excipient. In one embodiment, the enzyme powder is formed by spray drying (i.e., a spray-dried formulation). In one embodiment, the excipient is present in the enzyme powder in an amount ranging from about 95 wt% to about 25 wt% of the enzyme powder. In a further aspect, the excipient is an oligosaccharide excipient. In a further aspect, the oligosaccharide excipient has a number average molecular weight of at least about 1250 and a weight average molecular weight of at ieast about 9000, and optionally at least one surfactant.

The at least one enzyme can be any of the CE-7 carbohydrate esterases described herein or can be any of the CE-7 carbohydrate esterases described in co-owned, copending Published U.S. Patent Application No. 2008/0176299 (incorporated herein by reference in its entirety). In some embodiments, the at least one enzyme is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, and 25. in one embodiment, the at least one enzyme comprises an amino acid sequence substantially similar to SEQ ID NOs: 6, 7, 19, or 20. In a preferred embodiment, the at least one enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 19, and 20. The at least one enzyme is present in the enzyme powder in an amount in a range of from about 5 weight percent (wt %) to about 75 wt% based on the dry weight of the enzyme powder. A preferred weight percent range of the enzyme in the enzyme powder is from about 10 wt% to 50 wt%, and a more preferred weight percent range of the enzyme in the enzyme powder is from about 20 wt% to 33 wt%.

In one embodiment, the enzyme powder further comprises an excipient. In one aspect, the excipient is provided in an amount in a range of from about 95 wt% to about 25 wt% based on the dry weight of the enzyme powder. A preferred wt % range of excipient in the enzyme powder is from about 90 wt% to 50 wt%, and a more preferred wt % range of excipient in the enzyme powder is from about 80 wt% to 67 wt%.

In yet a further embodiment, the excipient is at least one oligosaccharide excipieint. !n still a further embodiment, at least one oligosaccharide excipient has a number average molecular weight of at least about 1250 and a weight average molecular weight of at least about 9000. In some embodiments, the oligosaccharide excipient has a number average molecular weight of at least about 1700 and a weight average molecular weight of at least about 15000. Specific oligosaccharides useful in the present invention include, but are not limited to, maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, insulin, Sevan, graminan, amylopectin, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose, and mixtures thereof. In a preferred embodiment, the oligosaccharide excipient is maltodextrin. Oligosaccharide-based excipients useful in the present invention may include, but are not limited to, water-soluble non-ionic cellulose ethers, such as hydroxymethyl-celluiose and hydroxypropylmethylcellulose, and mixtures thereof. In yet a further embodiment, the excipient is selected from, but not limited to, one or more of the following compounds: trehalose, lactose, sucrose, mannitol. sorbitol, glucose, celiobiose, α-cyciodextrin, carboxymethylcellulose.

In some embodiments, the formulation used to prepare the enzyme powder optionally comprises at least one surfactant. In a preferred aspect, at feast one surfactant is present. Useful surfactants may include, but are not limited to, ionic and nonionic surfactants or wetting agents, such as ethoxylated castor oil, polyglycotyzed giycerides, acetylated monoglycerides, sorbitan fatty acid esters, poloxamers, polyoxyethylene sorbitan fatty acid esters, polyoxyethyiene derivatives, monoglycerides or ethoxylated derivatives thereof, digSycerides or polyoxyethylene derivatives thereof, sodium docusate, sodium laurylsulfate, cholic acid or derivatives thereof, lecithins, phospholipids, block copolymers of ethylene glycol and propylene glycol, and non-ionic organosilicones. Preferably, the surfactant is a polyoxyethylene sorbitan fatty acid ester, with poiysorbate 80 being more preferred.

When part of the formulation used to prepare the enzyme powder, the surfactant is present in an amount in a range of from about 5 wt% to 0.1 wt% based on the weight of protein present in the enzyme powder, preferably from about 2 wt% to 0.5 wt% based on the weight of protein present in the enzyme powder. In a preferred embodiment, the enzyme powder/formuiation is formed by spray drying.

The formulation used to prepare the enzyme powder may additionally comprise one or more buffers (e.g., sodium and/or potassium salts of bicarbonate, citrate, acetate, phosphate, pyrophosphate, methylphosphonate, succinate, malate, fumarate, tartrate, or maleate), and an enzyme stabilizer (e.g., ethylenediaminetetraacetic acid, (I hydroxyethylidene)bisphosphonic acid).

Spray-drying of the formulation to form the enzyme powder is carried out, for example, as described generally in Spray _, Drying Handbook, 5 ^th ed., K. Masters, John Wiley & Sons, Inc., NY, N.Y. (1991), and in PCT Patent Publication Nos. WO 97/41833 and WO 96/32149 to Platz, R. et a/..

In general spray drying consists of bringing together a highly dispersed liquid and a sufficient volume of hot air to produce evaporation and drying of the liquid droplets. Typically the feed is sprayed into a current of warm filtered air that evaporates the sotvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Those skilled in the art will appreciate that several different types of apparatus may be used to provide the desired product. For example, commercial spray dryers manufactured by Buchi Ltd. (Postfach, Switzerland) or GEA Niro Corp. (Copenhagen, Denmark) will effectively produce particles of desired size. It will further be appreciated that these spray dryers, and specifically their atomizers, may be modified or customized for specialized applications, such as the simultaneous spraying of two solutions using a double nozzle technique. More specifically, a water-in-oϋ emulsion can be atomized from one nozzle and a solution containing an anti- adherent such as mannitol can be co-atomized from a second nozzle, in other cases it may be desirable to push the feed solution though a custom designed nozzle using a high pressure liquid chromatography (HPLC) pump. Provided that microstructures comprising the correct morphology and/or composition are produced the choice of apparatus is not critical and would be apparent to the skilled artisan in view of the teachings herein.

The temperature of both the inlet and outlet of the gas used to dry the sprayed material is such that it does not cause degradation of the enzyme in the sprayed material. Such temperatures are typically determined experimentally, although generally, the inlet temperature will range from about 50 ⁰ C to about 225 ⁰ C, while the outlet temperature will range from about 30 ⁰ C to about 150 ⁰ C. Preferred parameters include atomization pressures ranging from about 20-150 psi (0.14 MPa - 1.03 MPa), and preferably from about 30- 40 to 100 psi (0.21-0.28 MPa to 0.69 MPa). Typically the atomization pressure employed will be one of the following (MPa) 0.14, 0.21 , 0.28, 0.34, 0.41, 0.48, 0.55, 0.62, 0.69, 0.76, 0.83 or above.

The enzyme powder or a formulation of the enzyme powder in carboxylic acid ester substantially retains its enzymatic activity for an extended period of time when stored at ambient temperature. The enzyme powder or a formulation of the enzyme powder in carboxylic acid ester substantially retains its enzymatic activity at elevated temperatures for short periods of time. In one embodiment, "substantially retains its enzymatic activity" is meant that the enzyme powder or a formulation of the enzyme powder in carboxylic acid ester retains at least about 75 percent of the enzyme activity of the enzyme in the enzyme powder or a formulation of the enzyme powder after an extended storage period at ambient temperature and/or after a short storage period at an elevated temperature (above ambient temperature) in a formulation comprised of a carboxylic acid ester and the enzyme powder as compared to the initial enzyme activity of the enzyme powder prior to the preparation of a formulation comprised of the carboxylic acid ester and the enzyme powder. The extended storage period is a period of time of from about one year to about two years at ambient temperature. In one embodiment, the short storage period is at an elevated temperature is a period of time of from when the formulation comprised of a carboxylic acid ester and the enzyme powder is produced at 40 ⁰ C to about eight weeks at 40 ⁰ C. In another embodiment, the elevated temperature is in a range of from about 30 ⁰ C to about 52 ⁰ C. In a preferred embodiment, the elevated temperature is in a range of from about 30 ⁰ C to about 40 ⁰ C.

In some embodiments, the enzyme powder has at least 75 percent of the enzyme activity of the at least one enzyme after eight weeks storage at 40 ⁰ C in a formulation comprised of a carboxylic acid ester and the enzyme powder as compared to the initial enzyme activity of the enzyme powder prior to the preparation of a formulation comprised of the carboxylic acid ester and the enzyme powder at 40 ⁰ C. In other embodiments, the enzyme powder has at least 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of the enzyme activity of the at least one enzyme after eight weeks storage at 40 ⁰ C in a formulation comprised of a carboxylic acid ester and the enzyme powder as compared to the initial enzyme activity of the enzyme powder prior to the preparation of a formulation comprised of the carboxylic acid ester and the enzyme powder at 40 ⁰ C. Preferably, perhydrolysis activity is measured as described in Example 8-13, infra, but any method of measuring perhydrolysis activity can be used in the practice of the present invention.

In some embodiments, further improvement in enzyme activity over the stated periods of time can be achieved by adding a buffer having a buffering capacity in a pH range of from about 5.5 to about 9.5 to the formulation comprised of the carboxylic acid ester and the spray-dried enzyme powder. Suitable buffer for use in the formulation may include, but is not limited to, sodium salt, potassium salt, or mixtures of sodium or potassium salts of bicarbonate, pyrophosphate, phosphate, methyiphosphonate, citrate, acetate, malate, fumarate, tartrate maleate or succinate. Preferred buffers for use in the formulation comprised of the carboxyiic acid ester and the spray-dried enzyme powder include the sodium salt, potassium salt, or mixtures of sodium or potassium salts of bicarbonate, pyrophosphate, phosphate, methylphosphoπate, citrate, acetate, malate, fumarate, tartrate maleate or succinate. In preferred embodiment, the buffer comprises the sodium and/or potassium salts of bicarbonate.

In embodiments where a buffer is present in the carboxyiic acid ester and enzyme powder formulation, the buffer may be present in an amount in a range of from about 0.01 wt% to about 50 wt% based on the weight of carboxyiic acid ester in the formulation comprised of carboxyiic acid ester and enzyme powder. The buffer may be present in a more preferred range of from about 0.10 % to about 10 % based on the weight of carboxyiic acid ester in the formulation comprised of carboxyiic acid ester and enzyme powder. Further, in these embodiments, the comparison between perhydrolysis activity of the enzyme is determined as between an enzyme powder which retains at least 75 percent of the perhydrolysis activity of the at least one enzyme after eight weeks storage at 40 ⁰ C in a formulation comprised of a carboxyiic acid ester, a buffer having a buffering capacity in a pH range of from about 5.5 to about 9.5, and the enzyme powder as compared to the initial perhydrolysis activity of the enzyme powder prior to the preparation of a formulation comprised of the carboxylfc acid ester, the buffer having a buffering capacity in a pH range of from about 5.5 to about 9.5, and the enzyme powder.

It is intended that the enzyme powder be stored as a formulation in the organic compound that is a substrate for the at least one enzyme, such as triacetin. In the absence of added hydrogen peroxide, triacetin is normally hydrolyzed in aqueous solution by a CE-7 carbohydrate esterase to produce diacetin and acetic acid, and the production of acetic acid results in a decrease in the pH of the reaction formulation. One requirement for long term storage stability of the enzyme in triacetin is that there is not a significant reaction of the triacetin with any water that might be present in the triacetin; the specification for water content in one commercial triacetin (supplied by Tessenderlo Group, Brussels, Belgium) is 0.03 wt% water (300 ppm). Any hydrolysis of triacetin that occurs during storage of the enzyme in triacetin would produce acetic acid, which could result in a decrease in activity or inactivation of the CE-7 perhydrolases; the perhydrolases are typically inactivated at or below a pH of 5.0 (see U.S. Patent Application Publication No. 2009/0005590 to DiCosimo, R., et a/.). The excipient selected for use in the present application must provide stability of the enzyme in the organic substrate for the enzyme under conditions where acetic acid might be generated due to the presence of low concentrations of water in the formulation.

Suitable Reaction Conditions for the Enzyme-cataiyzed Preparation of Peroxycarboxylic acids from CarboxySic Acid Esters and Hydrogen Peroxide

In one aspect of the invention, a process is provided to produce an aqueous formulation comprising a peroxycarboxylic acid by reacting carboxylic acid esters with a source of peroxygen (including, but not limited to, hydrogen peroxide, sodium perborate or sodium percarbonate) in the presence of an enzyme catalyst having perhydrolysis activity. In one embodiment, the enzyme catalyst comprises at least one enzyme having perhydrolysis activity, wherein said enzyme is structurally classified as a member of the CE-7 carbohydrate esterase family (CE-7; see Coutinho, P.M., and Henrissat, B., supra). In another embodiment, the perhydrolase catalyst is structurally classified as a cephalosporin C deacetylase. In another embodiment, the perhydrolase catalyst is structurally classified as an acetyl xylan esterase.

In one embodiment, the perhydroiase catalyst comprises an enzyme having perhydrolysis activity and a signature motif comprising: a) an RGQ motif at amino acid residues 118-120; b) a GXSQG motif at amino acid residues 179-183; and c) an HE motif at amino acid residues 298-299 when aligned to reference sequence SEQ ID NO:1 using CLUSTALW. In a further embodiment, the signature motif additional comprises a fourth conserved motif defined as an LXD motif at amino acid residues 267- 269 when aligned to reference sequence SEQ ID NO:1 using CLUSTALW. In another embodiment, the perhydrolase catalyst comprises an enzyme having the present signature motif and at least 30% amino acid to SEQ ID NO:1.

In another embodiment, the perhydroiase catalyst comprises an enzyme having perhydrolase activity selected from the group consisting of SEQ ID

NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 19, 20, 21 , 22, 23, . 24, and 25.

In another embodiment, the perhydrolase catalyst comprises an enzyme having at least 40% amino acid identity to a contiguous signature motif defined as SEQ ID NO:18 wherein the conserved motifs described above (i.e., RGQ, GXSQG, and HE, and optionally, LXD) are conserved.

In another embodiment, the perhydrolase catalyst comprises an enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 19, 20, 21 , 22, 23, 24, and 25, wherein said enzyme may have one or more additions, deletions, or substitutions so long as the signature motif is conserved and perhydrolase activity is retained.

Suitable carboxylic acid ester substrates may include esters having the following formula: (a) one or more esters having the structure

wherein X is an ester group of the formula R ₆ C(O)O;

R ₆ is a C1 to C7 linear, branched or cyclic hydrocarbyf moiety, optionally substituted with a hydroxy! group or C1 to C4 aikoxy group, wherein Re optionally comprises one or more ether linkages where R ₆ is C2 to C7; R ₅ is a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety optionally substituted with a hydroxyl group, wherein each carbon atom in R ₅ individually comprises no more than one hydroxyl group or no more than one ester group, and wherein R ₅ optionally comprises one or more ether linkages; m is 1 to the number of carbon atoms in R _5ι said one or more esters having solubility in water of at least 5 ppm at 25 ⁰ C; or

(b) one or more giycerides having the structure

wherein Ri is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R ₃ and R ₄ are individually H or RiC(O); or

(c) one or more esters of the formula

wherein Ri is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxy! or a C1 to C4 alkoxy group and R ₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkyπyl, aryi, alkylaryl, alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n , or (CH ₂ CH(CH ₃ )-O) _n H and n is 1 to 10; or

(d) one or more acetylated monosaccharides, acetylated disaccharides, or acetylated polysaccharides; or

(e) any combination of (a) through (d). Examples of said carboxylic acid ester substrate may include monoacetin; triacetin; monopropionin; dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucose pentaacetate; xylose tetraacetate; acetyiated xylan; acetyiated xylan fragments; β-D-ribofuranose-1 ,2,3,5-tetraacetate; tri-O- acetyI-D-ga!actai; tri-O-acetyl-glucal; propylene glycol diacetate; ethylene glycol diacetate; monoesters or diesters of 1,2-ethanediol; 1 ,2-propanediol; 1 ,3-propanedio!; 1 ,2-butanediol; 1 ,3-butanediol; 2,3-butanediol; 1 ,4-butaπediol; 1 ,2-pentanediol; 2,5-pentanedioi; 1 ,6-peπtanediol, 1 ,2-hexanedioi; 2,5- hexaπediol; 1 ,6-hexanediol; or any combination thereof.

In a preferred embodiment, the carboxylic acid ester is a liquid substrate selected from the group consisting of monoacetin, diacetin, triacetin, and combinations (i.e., mixtures) thereof. The carboxylic acid ester is present in the reaction formulation at a concentration sufficient to produce the desired concentration of peroxycarboxylic acid upon enzyme-catalyzed perhydrolysis. The carboxylic acid ester need not be completely soluble in the reaction formulation, but has sufficient solubility to permit conversion of the ester by the perhydrolase catalyst to the corresponding peroxycarboxylic acid. The carboxylic acid ester is present in the reaction formulation at a concentration of 0.05 wt % to 40 wt % of the reaction formulation, preferably at a concentration of 0.1 wt % to 20 wt % of the reaction formulation, and more preferably at a concentration of 0.5 wt % to 10 wt % of the reaction formulation. The peroxygen source may include, but is not limited to, hydrogen peroxide, hydrogen peroxide adducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)) perborate salts and percarbonate salts. The concentration of peroxygen compound in the reaction formulation may range from 0.0033 wt % to about 50 wt %, preferably from 0.033 wt % to about 40 wt %, more preferably from 0.33 wt % to about 30 wt %.

Many perhydroiase catalysts (whole cells, permeabilized whole cells, and partially purified whole cell extracts) have been reported to have catalase activity (EC 1.11.1.6). Catalases catalyze the conversion of hydrogen peroxide into oxygen and water. In one aspect, the perhydrolysis catalyst lacks catalase activity, in another aspect, a catalase inhibitor is added to the reaction formulation. Examples of catalase inhibitors include, but are not limited to, sodium azide and hydroxylamine sulfate. One of skill in the art can adjust the concentration of catalase inhibitor as needed. The concentration of the catalase inhibitor typically ranges from 0.1 mM to about 1 M; preferably about 1 mM to about 50 mM; more preferably from about 1 mM to about 20 mM. In one aspect, sodium azide concentration typically ranges from about 20 mM to about 60 mM whiie hydroxylamine sulfate is concentration is typically about 0.5 mM to about 30 mM, preferably about 10 mM.

In another embodiment, the enzyme catalyst lacks significant cataiase activity or is engineered to decrease or eliminate catalase activity. The catalase activity in a host cell can be down-regulated or eliminated by disrupting expression of the gene(s) responsible for the cataiase activity using well known techniques including, but not limited to, transposon mutagenesis, RNA antisense expression, targeted mutagenesis, and random mutagenesis. In a preferred embodiment, the gene(s) encoding the endogenous catalase activity are down-regulated or disrupted (i.e., knocked-out). As used herein, a "disrupted" gene is one where the activity and/or function of the protein encoded by the modified gene is no longer present. Means to disrupt a gene are well-known in the art and may include, but are not limited to, insertions, deletions, or mutations to the gene so long as the activity and/or function of the corresponding protein is no longer present. In a further preferred embodiment, the production host is an E. coli production host comprising a disrupted catalase gene selected from the group consisting of katG and katE (see Published U.S. Patent Application No. 2008-0176299). In another embodiment, the production host is an E, coli strain comprising a down- regulation and/or disruption in both katG and a katE catalase genes.

The concentration of the catalyst in the aqueous reaction formulation depends on the specific catalytic activity of the catalyst, and is chosen to obtain the desired rate of reaction. The weight of catalyst in perhydrolysis reactions typically ranges from 0.0001 mg to 10 mg per mL of total reaction volume, preferably from 0.001 mg to 2.0 mg per mL. The catalyst may also be immobilized on a soluble or insoluble support using methods well-known to those skilled in the art; see for example, fmmobjljzation of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA; 1997. The use of immobilized catalysts permits the recovery and reuse of the catalyst in subsequent reactions. The enzyme catalyst may be in the form of whole microbial cells, permeabilized microbial ceiis, microbial cell extracts, partially- purified or purified enzymes, and mixtures thereof.

In one aspect, the concentration of peroxycarboxylic acid generated by the combination of chemical perhydrolysis and enzymatic perhydrolysis of the carboxylic acid ester is sufficient to provide an effective concentration of peroxycarboxylic acid for bleaching, stain removal, sanitization, odor reduction or disinfection at a desired pH. In another aspect, the present methods provide combinations of enzymes and enzyme substrates to produce the desired effective concentration of peroxycarboxylic acid, where, in the absence of added enzyme, there is a significantly lower concentration of peroxycarboxylic acid produced. Although there may in some cases be substantial chemical perhydrolysis of the enzyme substrate by direct chemical reaction of inorganic peroxide with the enzyme substrate, there may not be a sufficient concentration of peroxycarboxylic acid generated to provide an effective concentration of peroxycarboxylic acid in the desired applications, and a significant increase in total peroxycarboxylic acid concentration is achieved by the addition of an appropriate perhydrolase catalyst to the reaction formulation.

The concentration of peroxycarboxylic acid generated (such as peracetic acid) by the perhydrolysis of at least one carboxylic acid ester is at least about 20 ppm, preferably at least 100 ppm, more preferably at least about 200 ppm peroxycarboxyiic acid, more preferably at least 300 ppm, more preferably at least 500 ppm, more preferably at least 700 ppm, more preferably at least about 1000 ppm peroxycarboxylic acid, most preferably at least 2000 ppm peroxycarboxylic acid within 10 minutes, preferably within 5 minutes, of initiating the perhydrolysis reaction. The product formulation comprising the peroxycarboxylic acid may be optionally diluted with water, or a solution predominantly comprised of water, to produce a formulation with the desired lower concentration of peroxycarboxylic acid. In one aspect, the reaction time required to produce the desired concentration of peroxycarboxylic acid is not greater than about two hours, preferably not greater than about 30 minutes, more preferably not greater than about 10 minutes, and most preferably in about 5 minutes or less. In other aspects, a hard surface or inanimate object contaminated with a concentration of biological contaminant(s) is contacted with the peroxycarboxyiic acid formed in accordance with the processes described herein within about 5 minutes to about 168 hours of combining said reaction components, or within about 5 minutes to about 48 hours, or within about 5 minutes to 2 hours of combining said reaction components, or any such time interval therein.

In another aspect, the peroxycarboxyiic acid formed in accordance with the processes describe herein is used in a laundry care application wherein the peroxycarboxyiic acid is contacted with a textile to provide a benefit, such as disinfecting, sanitizing, bleaching, destaiπing, deodorizing/odor reduction or a combination thereof. The peroxycarboxyiic acid may be used in a variety of laundry care products including, but not limited to, textile pre-wash treatments, laundry detergents, stain removers, bleaching compositions, deodorizing compositions, and rinsing agents. !π one embodiment, the present process to produce a peroxycarboxyiic acid for a target surface is conducted in situ. In connection with the present systems and methods for laundry care where the peracid is generated for one or more of bleaching, stain removal, and odor reduction, the concentration of peracid generated (e.g., peracetic acid) by the perhydrolysis of at least one carboxylic acid ester may be at least about 2 ppm, preferably at least 20 ppm, preferably at least 100 ppm, and more preferably at least about 200 ppm peracid. In connection with the present systems and methods for laundry care where the peracid is generated for disinfection or sanitization, the concentration of peracid generated (e.g., peracetic acid) by the perhydrolysis of at least one carboxylic acid ester may be at least about 2 ppm, more preferably at least 20 ppm, more preferably at least 200 ppm, more preferably at least 500 ppm, more preferably at least 700 ppm, more preferably at least about 1000 ppm peracid, most preferably at least 2000 ppm peracid within 10 minutes, preferably within 5 minutes, and most preferably within 1 minute of initiating the perhydrolysis reaction. The product mixture comprising the peracid may be optionally diluted with water, or a solution predominantly comprised of water, to produce a mixture with the desired lower concentration of peracid. In one aspect of the present methods and systems, the reaction time required to produce the desired concentration of peracid is not greater than about two hours, preferably not greater than about 30 minutes, more preferably not greater than about 10 minutes, even more preferably not greater than about 5 minutes, and most preferably in about 1 minute or less.

The temperature of the reaction is chosen to control both the reaction rate and the stability of the enzyme catalyst activity. The temperature of the reaction may range from just above the freezing point of the reaction formulation (approximately 0 ⁰ C) to about 95 ⁰ C, with a preferred range of 5 ⁰ C to about 75 ⁰ C, and a more preferred range of reaction temperature of from about 5 ⁰ C to about 55 ⁰ C. The pH of the final reaction formulation containing peroxycarboxylic acid is from about 2 to about 9, preferably from about 3 to about 8, more preferably from about 5 to about 8, even more preferably about 5.5 to about 8, and yet even more preferably about 6.0 to about 7.5. In another embodiment, the pH of the reaction formulation is acidic (pH <7). The pH of the reaction, and of the final reaction formulation, may optionally be controlled by the addition of a suitable buffer, including, but not limited to, phosphate, pyrophosphate, bicarbonate, acetate, or citrate. The concentration of buffer, when employed, is typically from 0.1 mM to 1.0 M, preferably from 1 mM to 300 mM, most preferably from 10 mM to 100 mM. In another aspect, the enzymatic perhydrolysis reaction formulation may contain an organic solvent that acts as a dispersant to enhance the rate of dissolution of the carboxylic acid ester in the reaction formulation. Such solvents include, but are not limited to, propylene glycol methyl ether, acetone, cyclohexanone, diethylene glycol butyl ether, tripropylene glycol methyl ether, diethylene glycol methyl ether, propylene glycol butyl ether, dipropylene glycol methyl ether, cycSohexanol, benzyl alcohol, isopropanol, ethanol, propylene glycol, and mixtures thereof.

In another aspect, the enzymatic perhydrolysis product may contain additional components that provide desirable functionality. These additional components include, but are not limited to, buffers, detergent builders, thickening agents, emulsifiers, surfactants, wetting agents, corrosion inhibitors (such as benzotriazole), enzyme stabilizers, and peroxide stabilizers (e.g., metal ion chelating agents). Many of the additional components are welt known in the detergent industry (see, for example, U.S. Patent 5,932,532; hereby incorporated by reference). Examples of emulsifiers include, but are not limited to, polyvinyl alcohol or polyvinylpyrrolidone. Examples of thickening agents include, but are not limited to, LAPONITE ^® RD, corn starch, PVP, CARBOWAX ^® , CARBOPOL ^® CABOSI L ^® , polysorbate 20, PVA, and lecithin. Examples of buffering systems include, but are not limited to, sodium phosphate monobasic/sodium phosphate dibasic; sulfamic acid/triethanoiamine; citric acid/triethanolamine; tartaric acid/triethanolamine; succinic acid/triethanolamine; and acetic acid/triethanolamine. Examples of surfactants include, but are not limited to, a) non-ionic surfactants such as block copolymers of ethylene oxide or propylene oxide, ethoxylated or propoxylated linear and branched primary and secondary alcohols, and aliphatic phosphine oxides; b) cationic surfactants such as quaternary ammonium compounds, particularly quaternary ammonium compounds having a C8-C20 alkyl group bound to a nitrogen atom additionally bound to three C1- C2 alkyl groups; c) anionic surfactants such as alkane carboxylic acids (e.g., C8-C20 fatty acids), alkyl phosphonates, alkane sulfonates (e.g., sodium dodecylsulphate "SDS") or linear or branched alkyi benzene sulfonates, aikene sulfonates; and d) amphoteric and zwitterionic surfactants such as amiπocarboxylic acids, aminodicarboxylic acids, alkybetaines, and mixtures thereof. Additional components may include fragrances, dyes, stabilizers of hydrogen peroxide (e.g., metal chelators such as 1-hydroxyethylidene -1 ,1- diphosphonic acid (DEQUEST ^® 2010, Solutia Inc., St. Louis, MO and ethylenediaminetetraacetic acid (EDTA)), TURPINAL ^® SL (CAS# 2809-21-4), DEQUEST ^® 0520, DEQUEST ^® 0531 , stabilizers of enzyme activity (e.g., polyethylene glycol (PEG)), and detergent builders.

In Situ Production of Peroxycarboxyiic acids using a Perhydrplase Catalyst

Cephalosporin C deacetylases (E. C. 3.1.1.41 ; systematic name cephalosporin C acetylhydrolases; CAHs) are enzymes having the ability to hydrolyze the acetyl ester bond on cephalosporins such as cephalosporin C, 7- aminocephalosporanic acid, and 7-(thiophene-2-acetamido)cephafosporanic acid (Abbott, B. and Fukuda, D., Appl. Microbiol. 30(3):413-419 (1975)). CAHs belong to a larger family of structurally related enzymes referred to as the carbohydrate esterase family seven (CE-7; see Coutinho, P.M., Henrissat, B., supra).

The CE-7 family includes both CAHs and acetyl xylan esterases (AXEs; E. C. 3.1.1.72). CE-7 family members share a common structural motif and are quite unusual in that they typically exhibit ester hydrolysis activity for both acetylated xylooligosaccharides and acetylated cephalosporin C, suggesting that the CE-7 family represents a single class of proteins with a multifunctional deacetyiase activity against a range of small substrates (Vincent et a/., supra). Vincent et a/, describes the structural similarity among the members of this family and defines a signature sequence motif characteristic of the CE-7 family.

Members of the CE~7 family are found in plants, fungi (e.g., Cephalosporidium acremonium), yeasts (e.g., Rhodosporidium toruloides, Rhodotorula glutinis), and bacteria such as Thermoanaerobacterium sp.; Norcardia lactamdurans, and various members of the genus Bacillus (Politino et al., Appl. Environ. Microbiol., 63(12):4807-4811 (1997); Sakai et al., J. Ferment Bioeng, 85:53-57 (1998); Lorenz, W. and Wiegel, J., J. Bacteriol 179:5436-5441 (1997); Cardoza et al., Appl. Microbiol. Biotechnol., 54(3):406- 412 (2000); Mitsushima et al., supra] Abbott, B. and Fukuda, D., Appl. Microbiol. 30(3):413-419 (1975); Vincent et a/., supra ; Takami et a/., NAR, 28(21 ):4317-4331 (2000); Rey et al., Genome Biol., 5(10): article 77 (2004); Degrassi ef a/., Microbiology., 146:1585-1591 (2000); U.S. Patent 6,645,233;. U.S. Patent 5,281 ,525; U.S. Patent 5,338,676; and WO 99/03984.

WO2007/070609 and U.S. Patent Application Publication Nos. 2008/0176299, 2008/176783 and 2009/0005590 to DiCosimo et a/, disclose various enzymes structurally classified as CE-7 enzymes that have perhydrolysis activity suitable for producing efficacious concentrations of peroxycarboxylic acids from a variety of carboxylic acid ester substrates when combined with a source of peroxygen. Variant CE-7 enzymes having improved perhydrolysis activity are also described in a co-filed, co-owned, and copending U.S. Patent Application (Attorney Docket No. CL4392 US NA entitled "IMPROVED PERHYDROLASES FOR ENZYMATIC PERACID GENERATION", incorporated herein by reference in its entirety). The present method produces industrially-useful, efficacious concentrations of peroxycarboxyiic acids in situ under aqueous reaction conditions using the perhydrofase activity of an enzyme belonging to the CE-7 family of carbohydrate esterases.

HPLC Assay Method for Determining the Concentration of Peroxycarboxyiic acid and Hydrogen Peroxide.

A variety of analytical methods can be used in the present methods to analyze the reactaπts and products including, but not limited to, titration, high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectroscopy (MS), capillary electrophoresis (CE), the analytical procedure described by U. Karst et al., (Anal. Chem., 69(17):3623-3627 (1997)), and the 2,2 ^! -azino-bis (3-ethylbenzothazoline)-6-sulfonate (ABTS) assay (S. Minning, et ai, Analytica Chimica Acta 378:293-298 (1999) and WO 2004/058961 A1) as described in the present examples.

Determination of Minimum Biocidal Concentration of Peroxycarboxyiic acids

The method described by J. Gabriejson, et ai. [J. Microbiol. Methods 50: 63-73 (2002)) can be employed for determination of the Minimum Biocidal Concentration (MBC) of peroxycarboxyiic acids, or of hydrogen peroxide and enzyme substrates. The assay method is based on XTT reduction inhibition, where XTT ((2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5- [(phenylamino)carbonyl]~2H-tetrazoiium, inner salt, monosodium salt) is a redox dye that indicates microbial respiratory activity by a change in optical density (OD) measured at 490 πm or 450 nm. However, there are a variety of other methods available for testing the activity of disinfectants and antiseptics including, but not limited to, viable plate counts, direct microscopic counts, dry weight, turbidity measurements, absorbance, and bioluminescence (see, for example Brock, Semour S., Disinfection, Sterilization, and Preservation, 5 ^th edition, Lippincott Williams & Wilkins, Philadelphia, PA, USA; 2001). Uses of Enzymaticaily-Prepared Peroxycarboxylic acid Compositions

The enzyme catalyst-generated peroxycarboxylic acid produced according to the present method can be used in a variety of hard surface/inanimate object applications for reduction of concentrations of biological contaminants, such as decontamination of medical instruments (e.g., endoscopes), textiles (e.g., articles of clothing, garments, carpets), food preparation surfaces, food storage and food-packaging equipment, materials used for the packaging of food products, chicken hatcheries and grow-out facilities, animal enclosures, and spent process waters that have microbial and/or virucidal activity. The enzyme-generated peroxycarboxylic acids may be used in formulations designed to inactivate prions (e.g., certain proteases) to additionally provide biocidal activity. In a preferred aspect, the present peroxycarboxylic acid compositions are particularly useful as a disinfecting agent for non-autoclavable medical instruments and food packaging equipment. As the peroxycarboxylic acid-containing formulation may be prepared using GRAS or food-grade components (enzyme, enzyme substrate, hydrogen peroxide, and buffer), the enzyme-generated peroxycarboxylic acid may also be used for decontamination of anima! carcasses, meat, fruits and vegetables, or for decontamination of prepared foods. The enzyme-generated peroxycarboxylic acid may be incorporated into a product whose final form is a powder, liquid, gel, film, solid or aerosol. The enzyme-generated peroxycarboxylic acid may be diluted to a concentration that still provides an efficacious decontamination.

The compositions comprising an efficacious concentration of peroxycarboxylic acid can be used to disinfect or sanitize surfaces and/or objects contaminated (or suspected of being contaminated) with biological contaminants by contacting the surface or object with the products produced by the present processes. As used herein, "contacting" refers to placing a disinfecting composition comprising an effective concentration of peroxycarboxylic acid in contact with the surface or inanimate object suspected of contamination with a biological contaminant for a period of time sufficient to clean and disinfect. Contacting includes spraying, treating, immersing, flushing, pouring on or in, mixing, combining, painting, coating, applying, affixing to and otherwise communicating a peroxycarboxylic acid solution or composition comprising an efficacious concentration of peroxycarboxylic acid, or a solution or composition that forms an efficacious concentration of peroxycarboxylic acid, with the surface or inanimate object suspected of being contaminated with a concentration of a microbial population. The disinfectant compositions may be combined with a cleaning composition to provide both cleaning and disinfection. Alternatively, a cleaning agent (e.g., a surfactant or detergent) may be incorporated into the formulation to provide both cleaning and disinfection in a single composition, The compositions comprising an efficacious concentration of peroxycarboxylic acid can also contain at least one additional antimicrobial agent, combinations of prion-degrading proteases, a virucide, a sporicide, or a biocide. Combinations of these agents with the peroxycarboxylic acid produced by the claimed processes can provide for increased and/or synergistic effects when used to clean and disinfect surfaces and/or objects contaminated (or suspected of being contaminated) with biological contaminants. Suitable antimicrobial agents include carboxylic esters (e.g., p- hydroxy aikyi benzoates and alkyi cinnamates); sulfonic acids (e.g., dodecylbenzene sulfonic acid); iodo-compounds or active halogen compounds (e.g., elemental halogens, halogen oxides (e.g., NaOCl ₁ HOCI, HOBr, CIO ₂ ), iodine, interhalides (e.g., iodine monochloride, iodine dichioride, iodine trichloride, iodine tetrachloride, bromine chioride, iodine monobromide, or iodine dibromide), polyhalides, hypochlorite salts, hypochlorous acid, hypobromite salts, hypobromous acid, chloro- and bromo-hydantoins, chlorine dioxide, and sodium chlorite); organic peroxides including benzoyl peroxide, alkyl benzoyl peroxides, ozone, singlet oxygen generators, and mixtures thereof; phenolic derivatives (e.g., ophenyl phenol, o-benzyl-p-chlorophenol, ferf-amyl pheno! and C ₁ -C ₆ aikyl hydroxy benzoates); quaternary ammonium compounds (e.g., alkyldimethyl benzyl ammonium chloride, diaikyldimethyl ammonium chloride and mixtures thereof); and mixtures of such antimicrobial agents, in an amount sufficient to provide the desired degree of microbial protection. Effective amounts of antimicrobial agents include about 0.001 wt% to about 60 wt% antimicrobial agent, about 0.01 wt% to about 15 wt% antimicrobial agent, or about 0.08 wt% to about 2.5 wt% antimicrobial agent.

In one aspect, the peroxycarboxylic acids formed by the present process can be used to reduce the concentration of biological contaminants (such as a viable microbial population) when applied on and/or at a locus. As used herein, a "locus" of the invention comprises part or all of a target surface suitable for disinfecting or bleaching. Target surfaces include all surfaces that can potentially be contaminated with biologicai contaminants. Non-limiting examples include equipment surfaces found in the food or beverage industry (such as tanks, conveyors, floors, drains, coolers, freezers, equipment surfaces, walls, valves, belts, pipes, drains, joints, crevasses, combinations thereof, and the like); building surfaces (such as walls, floors and windows); πon~food-industry related pipes and drains, including water treatment facilities, pools and spas, and fermentation tanks; hospital or veterinary surfaces (such as walls, floors, beds, equipment (such as endoscopes), clothing worn in hospital/veterinary or other healthcare settings, including clothing, scrubs, shoes, and other hospital or veterinary surfaces); restaurant surfaces; bathroom surfaces; toilets; clothes and shoes; surfaces of barns or stables for livestock, such as poultry, cattle, dairy cows, goats, horses and pigs; hatcheries for poultry or for shrimp; and pharmaceutical or biopharmaceutical surfaces {e.g., pharmaceutical or biopharmaceutical manufacturing equipment, pharmaceutical or biopharmaceutical ingredients, pharmaceutical or biopharmaceutical excipieπts). Additional hard surfaces may also include food products, such as beef, poultry, pork, vegetables, fruits, seafood, combinations thereof, and the like. The locus can also include water absorbent materials such as infected linens or other textiles. The locus also includes harvested plants or plant products including seeds, conns, tubers, fruit, and vegetables, growing plants, and especially crop growing plants, including cereals, leaf vegetables and salad crops, root vegetables, legumes, berried fruits, citrus fruits and hard fruits.

Non-limiting examples of hard surface materials are metals (e.g., steel, stainless steel, chrome, titanium, iron, copper, brass, aluminum, and alloys thereof), minerals (e.g. , concrete), polymers and plastics (e.g., polyolefins, such as polyethylene, polypropylene, polystyrene, poly(meth)acrylate, polyacryloπitrile, polybutadiene, poly(acrylonitrile, butadiene, styrene), po!y(acryloπitrife, butadiene), acrylonitrile butadiene; polyesters such as polyethylene terephthalate; and polyamides such as nylon). Additional surfaces may include brick, tile, ceramic, porcelain, wood, vinyl, linoleum, and carpet.

The peroxycarboxylic acids formed by the present process may be used to provide a benefit to an article of clothing or a textile including, but not limited to, bleaching, odor reduction, stain removal, and disinfection. The peroxycarboxylic acids formed by the present process may be used in any number of laundry care products including, but not limited to, textile/clothing pre-wash treatments, laundry detergents, stain removers, bleaching compositions, deodorizing compositions, and rinsing agents.

In the context of laundry care applications, the term "contacting an article of clothing or textile" means that the article of clothing or textile is exposed to a formulation disclosed herein. To this end, there are a number of formats the formulation may be used to treat articles of clothing or textiles including, but not limited to, liquid, solids, gel, paste, bars, tablets, spray, foam, powder, or granules and can be delivered via hand dosing, unit dosing, dosing from a substrate, spraying and automatic dosing from a laundry washing or drying machine. Granular compositions can also be in compact form; liquid compositions can also be in a concentrated form.

When the formulations disclosed herein are used in a laundry machine, the formulation can further contain components typical to laundry detergents. For example, typical components included, but are not limited to, surfactants, bleaching agents, bleach activators, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents, softening agents, corrosion inhibitors, tarnish inhibitors, germicides, pH adjusting agents, non-builder alkalinity sources, chelating agents, organic and/or inorganic fillers, solvents, hydrotropes, optical brighteners, dyes, and perfumes.

The formulations disclosed herein can also be used as detergent additive products in solid or liquid form. Such additive products are intended to supplement or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process.

Recombinant Microbial Expression The genes and gene products of the instant sequences may be produced in heterologous host cells, particularly in the cells of microbial hosts. Preferred heterologous host cells for expression of the instant genes and nucleic acid molecules are microbial hosts that can be found within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances. For example, it is contemplated that any of bacteria, yeast, and filamentous fungi may suitably host the expression of the present nucleic acid molecules. The perhydrolase may be expressed iπtracellulariy, extracellularly, or a combination of both intracellular^ and extraceliuiarly, where extracellular expression renders recovery of the desired protein from a fermentation product more facile than methods for recovery of protein produced by intracellular expression. Transcription, translation and the protein biosyπthetic apparatus remain invariant relative to the cellular feedstock used to generate cellular bϊomass; functional genes will be expressed regardless. Examples of host strains include, but are not limited to, bacterial, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida, Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus. In one embodiment, bacterial host strains include Escherichia, Bacillus, Kluyveromyces, and Pseudomonas. In a preferred embodiment, the bacterial host ceil is Escherichia colt.

Large-scale microbial growth and functional gene expression may use a wide range of simple or complex carbohydrates, organic acids and alcohols or saturated hydrocarbons, such as methane or carbon dioxide in the case of photosynthetic or chemoautotrophic hosts, the form and amount of nitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrient including small inorganic ions. The regulation of growth rate may be affected by the addition, or not, of specific regulatory molecules to the culture and which are not typically considered nutrient or energy sources.

Vectors or cassettes useful for the transformation of suitable host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell and/or native to the production host, although such control regions need not be so derived.

Initiation control regions or promoters which are useful to drive expression of the present cephalosporin C deacetylase coding region in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including, but not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1 , URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); A0X1 (useful for expression in Pichia); and lac, araB, let, trp, IP _L , IP _R , T7, tac, and trc (useful for expression in Escherichia colϊ) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus.

Termination control regions may also be derived from various genes native to the preferred host cell. In one embodiment, the inclusion of a termination control region is optional. In another embodiment, the chimeric gene includes a termination control region derived from the preferred host cell.

Industrial Production

A variety of culture methodologies may be applied to produce the perhydrolase catalyst. For example, large-scale production of a specific gene product overexpressed from a recombinant microbial host may be produced by batch, fed-batch, and continuous culture methodologies. Batch and fed-batch culturiπg methods are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Siπauer Associates, Inc., Suπderiaπd, MA (1989) and Deshpande, Mukuπd V., Appl. Biochem. Biotechnol., 36:227 (1992).

Commercial production of the desired perhydrolase catalyst may also be accomplished with a continuous culture. Continuous cultures are an open system where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in log phase growth. Alternatively, continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added and valuable products, byproducts or waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials.

Recovery of the desired perhydrolase catalysts from a batch fermentation, fed-batch fermentation, or continuous culture, may be accomplished by any of the methods that are known to those skilled in the art. For example, when the enzyme catalyst is produced iπtracellularly, the cell paste is separated from the culture medium by centrifugation or membrane filtration, optionally washed with water or an aqueous buffer at a desired pH, then a suspension of the cell paste in an aqueous buffer at a desired pH is homogenized to produce a cell extract containing the desired enzyme catalyst. The cell extract may optionally be filtered through an appropriate filter aid such as ceϋte or silica to remove cell debris prior to a heat-treatment step to precipitate undesired protein from the enzyme catalyst solution. The solution containing the desired enzyme catalyst may then be separated from the precipitated cell debris and protein by membrane filtration or centrifugation, and the resulting partially-purified enzyme catalyst solution concentrated by additional membrane filtration, then optionally mixed with an appropriate carrier (for example, maltodextrin, phosphate buffer, citrate buffer, or mixtures thereof) and spray-dried to produce a solid powder comprising the desired enzyme catalyst.

When an amount, concentration, or other value or parameter is given either as a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope be limited to the specific values recited when defining a range.

GENERAL METHODS The following examples are provided to demonstrate preferred aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples follow techniques to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skilt in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the presently disclosed methods and examples.

All reagents and materials were obtained from DIFCO Laboratories (Detroit, Ml), GIBCO/BRL (Gaithersburg, MD) ₁ TCI America (Portland, OR), Roche Diagnostics Corporation (indianapolis, IN) or Sigma/Aldrich Chemical Company (St. Louis, MO), unless otherwise specified.

The following abbreviations in the specification correspond to units of measure, techniques, properties, or compounds as follows: "sec" or "s" means second(s), "min" means minute(s), "h" or "hr" means hour(s), "μl_" means microliter(s), "mL" means milliliters), "L" means liter(s), "mM" means millimoSar, "M" means molar, "mmol" means miliimole(s), "ppm" means part(s) per million, "wf means weight, "wt%" means weight percent, "g" means gram(s), "mg" means miiligram(s), "μ9" means microgram(s), "ng" means nanogram(s), "g" means gravity, "HPLC" means high performance liquid chromatography, ^κ dd H ₂ O" means distilled and deioπized water, "dew" means dry celi weight, "ATCC" or "ATCC®" means the American Type Culture Collection (Manassas, VA), "U" means unit(s) of perhydrolase activity, "rpm" means revolution(s) per minute, "Tg" means glass transition temperature, and "EDTA" means ethylenediaminetetraacetic acid.

EXAMPLE 1 Construction of a katG Catalase Disrupted E. coli Strain

The coding region of the kanamycin resistance gene {kan\ SEQ ID NO:26) was amplified from the plasmid pKD13 (SEQ ID NO:27) by PCR (0.5 min at 94 °C, 0.5 miπ at 55 °C, 1 min at 70 ⁰ C, 30 cycles) using primers identified as SEQ ID NO:28 and SEQ ID NO:29 to generate the PCR product identified as SEQ ID NO:30. The katG nucleic acid sequence is provided as SEQ ID NO:31 and the corresponding amino acid sequence is SEQ iD NO:32. E. coli MG1655 (ATCC ^® 47076™) was transformed with the temperature- sensitive plasmid pKD46 (SEQ ID NO: 33), which contains the λ-Red recombinase genes (Datsenko and Wanner, (2000), PNAS USA 97:6640- 6645), and selected on LB-amp plates for 24 h at 30 ⁰ C. MG1655/pKD46 was transformed with 50-500 ng of the PCR product by electro porati on (BioRad Gene Puiser, 0.2 cm cuvette, 2.5 kV, 200 W, 25 μF), and selected on LB-kan plates for 24 h at 37 °C. Several colonies were streaked onto LB-kan plates and incubated overnight at 42 °C to cure the pKD46 plasmid. Colonies were checked to confirm a pheπotype of kaπR/ampS. Genomic DNA was isolated from several colonies using the PUREGENE ^® DNA purification system (Gentra Systems, Minneapolis, MN), and checked by PCR to confirm disruption of the katG gene using primers identified as SEQ ID NO:34 and SEQ ID NO:35. Several /caK3-disrupted strains were transformed with the temperature- sensitive plasmid pCP20 (SEQ ID NO:36), which contains the FLP recombinase, used to excise the kan gene, and selected on LB-amp plates for 24 h at 37 ⁰ C. Several colonies were streaked onto LB plates and incubated overnight at 42 °C to cure the pCP20 plasmid. Two colonies were checked to confirm a phenotype of kanS/ampS, and called MG1655 KatG1 and MG1655 KatG2.

EXAMPLE 2 Construction of a katE Catalase Disrupted E. coli Strain

The kanamycin resistance gene (SEQ ID NO:26) was amplified from the piasmid pKD13 (SEQ ID NO:27) by PCR (0.5 min at 94 ⁰ C, 0.5 min at 55 ⁰ C ₁ 1 min at 70 °C, 30 cycles) using primers identified as SEQ ID NO:37 and SEQ ID NO:38 to generate the PCR product identified as SEQ ID NO:39. The katE nucleic acid sequence is provided as SEQ ID NO:40 and the corresponding amino acid sequence is SEQ ID NO:41. E. coli MG 1655 (ATCC ^® 47076™) was transformed with the temperature-sensitive piasmid pKD46 (SEQ ID NO: 33), which contains the λ-Red recombinase genes, and selected on LB-amp plates for 24 h at 30 ⁰ C. MG1655/pKD46 was transformed with 50-500 ng of the PCR product by electroporation (BioRad Gene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W, 25 μF), and selected on LB-kan plates for 24 h at 37 ⁰ C. Several colonies were streaked onto LB-kan plates and incubated overnight at 42 ⁰ C to cure the pKD46 piasmid. Colonies were checked to confirm a phenotype of kanR/ampS. Genomic DNA was isolated from several colonies using the PUREGENE ^® DNA purification system, and checked by PCR to confirm disruption of the katE gene using primers identified as SEQ ID NO: 42 and SEQ ID NO: 43. Several /cafE-disrupted strains were transformed with the temperature-sensitive piasmid pCP20 (SEQ ID NO: 36), which contains the FLP recombinase, used to excise the kan gene, and selected on LB-amp plates for 24 h at 37 ⁰ C. Several colonies were streaked onto LB plates and incubated overnight at 42 ⁰ C to cure the pCP20 pSasmid. Two colonies were checked to confirm a phenotype of kanS/ampS, and called MG 1655 KatE1 and MG1655 KatE2. EXAMPLE 3 Construction of a katG catalase and katE Catalase Disrupted E. coli Strain

(KLP 18)

The kanamycin resistance gene (SEQ ID NO:26) was amplified from the plasmid pKD13 (SEQ ID NO:27) by PCR (0.5 min at 94 ⁰ C, 0.5 min at 55 ⁰ C, 1 min at 70 ⁰ C, 30 cycles) using primers identified as SEQ ID NO:37 and SEQ ID NO:38 to generate the PCR product identified as SEQ ID NO: 39. E. coli MG 1655 KatG1 (EXAMPLE 1) was transformed with the temperature-sensitive plasmid pKD46 (SEQ ID NO:33), which contains the λ-Red recombinase genes, and selected on LB-amp plates for 24 h at 30 ⁰ C. MG 1655

KatG1/pKD46 was transformed with 50-500 ng of the PCR product by electroporation (BioRad Gene Puiser, 0.2 cm cuvette, 2.5 kV, 200 W, 25 μF), and selected on LB-kan plates for 24 h at 37 ⁰ C. Several colonies were streaked onto LB-kan plates and incubated overnight at 42 ⁰ C to cure the pKD46 plasmid. Colonies were checked to confirm a phenotype of kanR/ampS. Genomic DNA was isolated from several colonies using the PUREGENE ^® DNA purification system, and checked by PCR to confirm disruption of the katE gene using primers identified as SEQ ID NO:42 and SEQ ID NO:43. Several /caflΞ-disrupted strains (Δ katE) were transformed with the temperature-sensitive plasmid pCP20 (SEQ ID NO:36), which contains the FLP recombinase, used to excise the kan gene, and selected on LB-amp plates for 24 h at 37 ⁰ C. Several colonies were streaked onto LB plates and incubated overnight at 42 ⁰ C to cure the pCP20 plasmid. Two colonies were checked to confirm a phenotype of kanS/ampS, and called MG 1655 KatG 1 KatE 18.1 and MG 1655 KatG1 KatE23. MG 1655 KatG 1 KatE 18.1 is designated E. coli KLP 18.

EXAMPLE 4

Cloning and Expression of Perhydrojase from Thermotoga neapolitana The coding region of the gene encoding acetyl xyian esterase from

Thermotoga neapolitana as reported in GENBANK ^® (accession number AE000512; region 80481-81458; SEQ ID NO:44) was synthesized using codons optimized for expression in E. coli (DNA 2.0, Menlo Park, CA). The coding region of the gene was subsequently amplified by PCR (0.5 min at 94 ⁰ C, 0.5 min at 55 ⁰ C, 1 min at 70 ⁰ C, 30 cycles) using primers identified as SEQ ID NO: 45 and SEQ ID NO: 46. The resulting nucleic acid product (SEQ !D NO: 47) was subcioned into pTrcHis2-TOPO ^® to generate the plasmid identified as pSW196. The plasmid pSW196 was used to transform E coli KLP18 (EXAMPLE 3) to generate the strain KLP18/pSW196. KLP18/pSW196 was grown in LB media at 37 ⁰ C with shaking up to OD ₆₀ oπm = 0.4-0.5, at which time IPTG was added to a final concentration of 1 mM, and incubation continued for 2-3 h. Cells were harvested by centrifugation and SDS-PAGE was performed to confirm expression of the perhydrolase at 20-40% of total soluble protein.

EXAMPLE 5 Cloning and Expression of Perhydrolase from Thermotoaa maritima MSB8 The coding region of the gene encoding acetyl xylan esterase from

Thermotoga maritima MSB8 as reported in GENBANK ^® (accession # NP_227893.1 ; SEQ ID NO: 48) was synthesized (DNA 2.0, Menlo Park, CA). The coding region of the gene was subsequently amplified by PCR (0.5 min @ 94 ⁰ C, 0.5 min @ 55 ⁰ C, 1 min @ 70 ⁰ C, 30 cycles) using primers identified as SEQ ID NO:49 and SEQ ID NO:50. The resulting nucleic acid product (SEQ ID NO:51) was cut with restriction enzymes Pst\ and Xba\ and subcioned between the Psti and Xba\ sites in pUC19 to generate the plasmid identified as pSW207. The piasmid pSW207 was used to transform E. coli KLP18 (EXAMPLE 3) to generate the strain identified as KLP18/pSW207. KLP18/pSW207 was grown in LB media at 37 ⁰ C with shaking up to OD ₆₀ oπm = 0.4-0.5, at which time IPTG was added to a final concentration of 1 mM, and incubation continued for 2-3 h. Cells were harvested by centrifugation and SDS-PAGE was performed to confirm expression of the perhydrolase enzyme at 20-40% of total soluble protein. EXAMPLE 6

Fermentation of E coli KLP18 Transformants Expressing Perhydroiase A fermentor seed culture was prepared by charging a 2-L shake flask with 0.5 L seed medium containing yeast extract (Amberex 695, 5.0 g/L), K ₂ HPO ₄ (10.0 g/L), KH ₂ PO ₄ (7.0 g/L), sodium citrate dihydrate (1.0 g/L), (NH ₄ ) ₂ SO ₄ (4.0 g/L), MgSO ₄ heptahydrate (1.0 g/L) and ferric ammonium citrate (0.10 g/L). The pH of the medium was adjusted to 6.8 and the medium was sterilized in the flask. Post sterilization additions included glucose (50 wt %, 10.0 mL) and 1 mL ampicϋlin (25 mg/mL) stock solution. The seed medium was inoculated with a 1-mL culture of E coli KLP18/pSW196 or E. coli

KLP18/pSW207 in 20% glycerol, and cultivated at 35 ⁰ C and 300 rpm. The seed culture was transferred at ca. 1-2 OD _550HfT i to a 14-L fermentor (Braun Biotech, Alientown, PA) with 8 L of medium at 35 ⁰ C containing KH ₂ PO ₄ (3.50 g/L), FeSO ₄ heptahydrate (0.05 g/L), MgSO ₄ heptahydrate (2.0 g/L), sodium citrate dihydrate (1.90 g/L), yeast extract (Amberex 695, 5.0 g/L),

Biospumex153K antifoam (0.25 mL/L, Cognis Corporation, Monheim, Germany), NaCI (1.0 g/L), CaCI ₂ dihydrate (10 g/L), and NIT trace elements solution (10 mL/L). The trace elements solution contained citric acid monohydrate (10 g/L), MnSO ₄ hydrate (2 g/L), NaCI (2 g/L), FeSO ₄ heptahydrate (0.5 g/L), ZnSO ₄ heptahydrate (0.2 g/L), CuSO ₄ pentahydrate (0.02 g/L) and NaMoO ₄ dihydrate (0.02 g/L). Post sterilization additions included glucose solution (50% w/w, 80.0 g) and ampicillin (25 mg/mL) stock solution (16.00 mL). Glucose solution (50% w/w) was used for fed batch. Glucose feed was initiated when glucose concentration decreased to 0.5 g/L, starting at 0.31 g feed/min and increasing progressively each hour to 0.36,

0.42, 0.49, 0.57, 0.66, 0.77, 0.90, 1.04, 1.21, 1.41 , and 1.63 g/min respectively; the rate remained constant afterwards. Glucose concentration in the medium was monitored and if the concentration exceeded 0.1 g/L the feed rate was decreased or stopped temporarily. Induction was initiated between OD ₅ 5θπ _m = 56 and OD ₆₆ o _nm - 80 with addition of 16 mL IPTG (0.5 M) for the various strains. The dissolved oxygen (DO) concentration was controlled at 25% of air saturation. The DO was controlled first by impeller agitation rate (400 to 1400 rpm) and later by aeration rate (2 to 10 slpm). The pH was controlled at 6.8. NH ₄ OH (29% w/w) and H ₂ SO ₄ (20% w/v) were used for pH control The head pressure was 0.5 bars. The cells were harvested by centrifugation 16 h post IPTG addition.

EXAMPLE 7

Preparation of Heat-Treated Cell Extracts of CE-7 Esterases/Perhydrolases

A cell extract of an E. coli transformaπt expressing perhydrolase from Thermotoga neapolitana (KLPi8/pSW196) or Thermotoga maritima MSB8 (KLP18/pSW207) was prepared by passing a suspension of eel! paste (20 wt % wet cell weight) in 0.05 M potassium phosphate buffer (pH 7.0) containing dithiothreitol (1 mM) twice through a French press having a working pressure of 16,000 psi (-110 MPa). The crude extract was then centrifuged at 20,000 x g to remove cellular debris, producing a clarified cell extract that was assayed for total soluble protein (Bicinchoniπic Acid Kit for Protein Determination, Sigma Aldrich catalog # BCA1-KT). The clarified Thermotoga maritima MSB8 or Thermotoga neapolitana perhydroiase-containing extract was heated for 20 min at 75 ⁰ C, followed immediately by cooling in an ice/water bath to 5 ^D C. The resulting mixture was centrifuged to remove precipitated protein, and the supernatant collected and assayed for total soluble protein as before. SDS- PAGE of the heat-treated supernatant indicated that the perhydrolase constituted at least ca. 90 % of the total soluble protein present in the supernatant.

EXAMPLE 8 Temperature Stability of T. neapolitana PerhydrolaserTrehalose Spray-Dried

Enzyme Powders

A set of ten aqueous mixtures were prepared that contained varying concentrations of the heat-treated ceil extract protein of E. coli KLP18/pSW196 (> 90 % T. neapolitana perhydrolase by PAGE), trehalose (Cargili), and, optionally, polysorbate 80 (p80) as surfactant in sodium bicarbonate buffer (50 mM, pH = 8.1) (Table 1). These solutions were spray-dried using a Buchi B- 290 glass-chamber spray dryer (inlet temperature = 170 ⁰ C, exit temperature = 90 ⁰ C, feed rate = 3 mL/min to 10 mL/min) to produce ten spray-dried enzyme powders; the weight percent protein in the powders was determined using the BCA (Bicinchoninic acid) protein assay, and the glass transition temperatures (Tg) of these powders were measured using modulated differential scanning calorimetry (Table 1 ).

Table 1. Composition of protein/excipient solutions used to produce T. neapolitana perhydrolase/trehalose spray-dried enzyme powders, and Tg of corresponding powders.

The spray-dried enzyme powders were stored in sealed vials at 40 ⁰ C and sampled at one-week intervals, and the samples assayed for the concentration of peracetic acid produced in 5 minutes in reactions containing T. neapolitana perhydrolase (50 μg protein/ml_), H ₂ O ₂ (100 mM), triacetin (100 mM) and TURPINAL ^® SL (500 ppm) in sodium bicarbonate buffer (50 mM, pH 7.2) at 25 ⁰ C, and analyzed for production of peracetic acid using a modification of the analytical method reported by Karst et al. (below). A sampie (0.040 ml_) of the reaction mixture was removed at a predetermined time (5 min) and immediately mixed with 0.960 ml_ of 5 mM phosphoric acid in water to terminate the reaction by adjusting the pH of the diluted sample to less than pH 4. The resulting solution was filtered using an ULTRAFREE ^® MC-filter unit (30,000 Normal Molecular Weight Limit (NMWL) ₁ Millipore Corp., Billerica, MA; cat # UFC3LKT 00) by centrifugation for 2 min at 12,000 rpm. An aliquot (0.100 mL) of the resulting filtrate was transferred to a 1.5-mL screw cap HPLC vial (Agilent Technologies, Palo Alto, CA; #5182- 0715) containing 0.300 mL of deionized water, then 0.100 mL of 20 mM MTS (methyl-p-toly! sulfide) in acetonttriie was added, the via! capped, and the contents briefly mixed prior to a 10 min incubation at ca. 25 °C in the absence of light. To the vial was then added 0.400 mL of acetonitrile and 0.100 mL of a solution of triphenylphosphine (TPP, 40 mM) in acetonitriie, the vial re-capped, and the resulting solution mixed and incubated at ca. 25 ⁰ C for 30 min in the absence of light. To the vial was then added 0.100 mL of 10 mM N,N-diethyl- m-toluamide (DEET; HPLC externa! standard) and the resulting solution analyzed by HPLC for MTSO (methyi-p-tolyl sulfoxide), the stoichiometric oxidation product produced by reaction of MTS with peracetic acid. A control reaction was run in the absence of added extract protein or triacetin to determine the rate of oxidation of MTS in the assay mixture by hydrogen peroxide, for correction of the rate of peracetic acid production for background MTS oxidation. HPLC method: Supelco Discovery C8 column (10-cm X 4.0- mm, 5 μm) (cat. #569422-U) with Supeico Supeiguard Discovery C8 precolumn (Sigma-Aldrich; catalog #59590-U); 10 microliter injection volume; gradient method with CH ₃ CN (Sigma-Aldrich; catalog #270717) and deionized water at 1.0 mL/min and ambient temperature.

Table 2. HPLC Gradient for analysis of peracetic acid.

The perhydrolytic activity of the T. neapolitana perhydrolase/trehaiose spray-dried powder was stable over eight weeks of storage at 40 ⁰ C (Table 2).

Table 3. Temperature stability of T. neapolitana perhydrolase/trehaiose spray- dried enzyme powders during storage at 40 ⁰ C. PAA (ppm) produced in 5 min at 25 ^C C by reaction of triacetin (100 mM) and H ₂ O ₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. neapolitana perhydroiase/ trehalose spray-dried powder (50 μg protein/mL) and TURPINAL ^® SL (500 ppm).

EXAMPLE 9

Temperature Stability of T. neapolitana Perhydroiase/Trehalose Spray-Dried Enzyme Powders in a Mixture of Enzyme Powder and Triacetin

The spray-dried enzyme powders prepared as described in Example 8 were evaluated for stability when stored for eight weeks at 40 ⁰ C as a mixture of the spray-dried powder in triacetin. Spray-dried enzyme powders were added to triacetiπ to produce a mixture containing 0.200 g of protein in 87.2 g of triacetiπ. The resulting mixtures were stored at 40 "C, and a 2.19 g sample of the well-stirred mixture was assayed weekly at 25 "C in a 100-mL reaction containing 100 mM hydrogen peroxide and TURPI NAL ^® SL (500 ppm) in 50 mM sodium bicarbonate buffer at pH 7.2, where the resulting concentration of triacetiπ and protein was 100 mM and 50 μg/mL, respectively. Comparison of the data in Table 4 with the data in Example 8, Table 3, demonstrates the instability of T. neapolitana perhydrolase/trehalose spray-dried enzyme powders when stored as a mixture with triacetin.

Table 4, Temperature stability of T. neapolitana perhydrolase/trehalose spray- dried enzyme powders during storage in a mixture of enzyme powder and triacetiπ at 40 ⁰ C. PAA (ppm) produced in 5 min at 25 ⁰ C by reaction of triacetiπ (100 mM) and H ₂ O ₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. neapolitana perhydrolase (50 μg protein/ml_) and TURPINAL ^® SL (500 ppm).

EXAMPLE 10 Temperature Stability of T. neapolitana Perhydrolase/Maltodextrin Spray-Dried

Enzyme Powder

An aqueous mixture was prepared containing heat-treated ceil extract protein of E, coli KLP18/pSW196 (34 g protein/L, > 90 % T. neapolitana perhydrolase by PAGE) and maltodextrin (66.7 g/L MALTRIN ^® M100 maltodextrin, 14.7 g/L MALTRIN ^® M250, 14.7 g/L MALTRIN ^® M040, Grain Processing Corporation, Muscatine, IA) as excipient in 50 mM sodium bicarbonate (pH 8.1). This solution was spray-dried using a spray dryer (GEA Niro, 3-ft diameter, iniet temperature = 226 ⁰ C, exit temperature - 76 ⁰ C, feed rate = 60 g/min) to produce a spray-dried enzyme powder; the weight percent protein in the powder (20.3 wt%) was determined using the BCA (Bicinchoninic acid) protein assay, and the glass transition temperature of this powder (Tg = 54 ⁰ C) was measured using modulated differential scanning calorimetry. This solution was spray-dried to produce a powder that was then tested for stability during storage at 40 ⁰ C for 9 weeks. The spray-dried enzyme powder (stored at 40 ⁰ C) was sampled at one-week intervals and assayed for activity using 50 μg protein/mL of T. neapolitana perhydrolase, H ₂ O ₂ (100 mM), triacetin (100 mM) and TURPINAL ^® SL (500 ppm) in 50 mM bicarbonate buffer (pH 7.2) at 25°C, and analyzed for production of peracetic acid using a modification of the analytical method reported by Karst et a/., supra. The perhydrolytic activity of the T. neapolitana perhydroiase/maitodextrin spray-dried powder was stable over eight weeks of storage at 40 ^D C (Table 5).

Table 5. Temperature stability of T. neapolitana perhydroiase/maitodextrin spray-dried enzyme powder during storage at 40 "C. PAA (ppm) produced in 5 min at 25 ⁰ C by reaction of triacetin (100 mM) and H ₂ O ₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. neapolitana perhydroiase (50 μg protein/mL) and TURPINAL ^® SL (500 ppm).

EXAMPLE 11

Temperature Stability of T. neapolitana perhydrolase/Maltodextrin Spray-Dried Enzyme Powder Stored in a Mixture of Enzyme Powder and Triacetin

The spray-dried enzyme powder prepared as described in Example 10 was evaluated for stability when stored for twenty-one weeks at 40 ⁰ C as a mixture of the spray-dried powder in triacetin. The spray-dried enzyme powder {1.235 g, 20.3 wt % protein) was added to 109 g of triacetin. The resulting mixture was stored at 40 ⁰ C, and a 2.19 g sample of the well-stirred mixture assayed in duplicate at 25 ^D C in a 100-mL reaction containing hydrogen peroxide (100 mM) and TURPINAL ^® SL (500 ppm) in 50 mM sodium bicarbonate buffer at pH 7.2, where the resulting concentration of triacetin and protein was 100 mM and 50 μg/mL, respectively. Comparison of the data in Table 6 with the data in Example 10, Table 5, demonstrates the stability of T. neapolitana perhydroiase/maltodextrin spray-dried enzyme powders when stored as a mixture with triacetin.

Table 6. Temperature stability of T. neapolitana perhydroiase/maltodextrin spray-dried enzyme powder during storage in a mixture of enzyme powder and triacetin at 40 ⁰ C. PAA (ppm) produced in 5 min at 25 ⁰ C by reaction of triacetin (100 mM) and H ₂ O ₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. neapolitana perhydrolase (50 μg protein/mL) and TURPINAL ^® SL (500 ppm).

time PAA (ppm) in 5 min

ND = a duplicate assay was not done

EXAMPLE 12

Temperature Stability of T. neapolitana Perhydroiase/Maltodextrin Spray-Dried Enzyme Powder Stored in a Mixture of Enzyme Powder, Sodium Bicarbonate and Triacetin

The spray-dried enzyme powder prepared as described in Example 10 was evaluated for stability when stored for 21 weeks at 40 ⁰ C as a mixture of the spray-dried powder in a mixture of triacetin and sodium bicarbonate. The spray-dried enzyme powder (0.988 g, 20.3 wt % protein) was added to a mixture of 87.2 g of triacetin and 16.8 g of sodium bicarbonate (Grade 3DF (powder), Church & Dwight). The resulting mixture was stored at 40 °C, and a 2.62 g sample of the weii-stirred mixture was assayed in duplicate at 25 ⁰ C in a 100-mL reaction containing hydrogen peroxide (100 mM) and TURPINAL ^® SL (500 ppm), where the resulting concentrations of triacetin, sodium bicarbonate and protein were 100 mM, 50 mM (pH 7.2) and 50 μg/mL, respectively. Comparison of the data in Table 7 with the data in Example 11, Table 6, demonstrates the stability of T, neapolitana perhydrolase/maltodextrin spray- dried enzyme powders when stored for twenty-one weeks at 40 ⁰ C as a mixture with triacetin and solid sodium bicarbonate is improved when compared to the stability of T. neapolitana perhydrolase/maitodextrin spray- dried enzyme powders when stored for twenty-one weeks at 40 ⁰ C as a mixture with triacetin alone. At the longer storage times, such as 21 weeks, the perhydrolase still retains ca. 100 % of initial activity in a mixture of triacetin and sodium bicarbonate.

Table 7. Temperature stability of T. neapolitana perhydrolase/maitodextrin spray-dried enzyme powder during storage in a mixture of enzyme powder, sodium bicarbonate and triacetin at 40 °C. PAA (ppm) produced in 5 min at 25 ⁰ C by reaction of triacetin (100 mM) and H ₂ O ₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. neapolitana perhydrolase (50 μg protein/mL) and TURPINAL ^® SL (500 ppm).

ND = a duplicate assay was not done

EXAMPLE 13 Temperature Stability of T. mahtlma Perhydrolase/Maltodextrin Spray-Dried

Enzyme Powder

An aqueous mixture was prepared containing heat-treated cell extract protein of E coli KLP18/pSW207 (21 g protein/L, > 90% T. maritime perhydrolase by PAGE) and maitodextrin (31 g/L maltodextrin DE 13-17 and 31 g/L maltodextrin DE 4-7, Aldrich) as excipient in 50 mM sodium bicarbonate (pH 8.1 ). This solution was spray-dried using a Buchi B-290 giass-chamber spray dryer (inlet temperature = 170 ⁰ C, exit temperature = 90 °C, feed rate = 4.5 mL/min) to produce a spray-dried enzyme powder; the weight percent protein in the powder (18.0 wt %) was determined using the BCA (Bicinchoninic acid) protein assay, and the giass transition temperature of this powder (Tg - 90 °C) was measured using modulated differential scanning calorimetry. This solution was spray-dried to produce a powder that was then tested for stability during storage at 40 ⁰ C for 7 weeks. The spray-dried enzyme powder (stored at 40 ⁰ C) was sampled at one-week intervals and assayed for activity using 50 μg protein/mL of T. maritima perhydrolase, H ₂ O ₂ (H ₂ O ₂ (100 mM)), triacetin (100 mM) and TURPINAL ^® SL (500 ppm) in 50 mM bicarbonate buffer (pH 7.2) at 25 ⁰ C, and analyzed for production of peracetic acid using a modification of the analytical method reported by Karst et a/., supra. The perhydrolytic activity of the T. maritima perhydrolase/maltodextrin spray-dried powder was stable over seven weeks of storage at 40 ^D C (Table 8).

Table 8. Temperature stability of T, maritima perhydrolase/maltodextrin spray- dried enzyme powder during storage at 40 ⁰ C, PAA (ppm) produced in 5 min at 25 ⁰ C by reaction of triacetin (100 mM) and H ₂ O ₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. maritima perhydrolase (50 μg protein/mL) and TURPINAL ^® SL (500 ppm).

EXAMPLE 14

Temperature Stability of 7 ^* . maritima Perhydroiase/Maltodextrin Spray-Dried Enzyme Powder Stored in a Mixture of Enzyme Powder and Triacetin

The spray-dried enzyme powder prepared as described in Example 13 was evaluated for stability when stored for seven weeks at 40 ⁰ C as a mixture of the spray-dried powder in triacetin. The spray-dried enzyme powder (0,556 g, 18.0 wt % protein) was added to 43.6 g of triacetin. The resulting mixture was stored at 40 °C, and a 2.21 g sample of the well-stirred mixture assayed in duplicate at 25 °C in a 100-mL reaction containing hydrogen peroxide (100 mM) and TURPINAL ^® SL (500 ppm) in 50 mM sodium bicarbonate buffer at pH 7.2, where the resulting concentration of triacetin and protein was 100 mM and 50 μg/mL, respectively. Comparison of the data in Table 9 with the data in Example 13, Table 8, demonstrates the stability of T. maritima perhydrolase/maltodextrin spray-dried enzyme powders when stored as a mixture with triacetin.

Table 9. Temperature stability of T. maritima perhydrolase/maltodextrin spray- dried enzyme powder during storage in a mixture of enzyme powder and triacetin at 40 ⁰ C. PAA (ppm) produced in 5 min at 25 ⁰ C by reaction of triacetin (100 mM) and H ₂ O ₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. maritima perhydrolase (50 μg protein/mL) and TURPINAL ^® SL (500 ppm).

EXAMPLE 15

Temperature Stability of T, maritima Perhydrolase/Maltodextrin Spray-Dried Enzyme Powder Stored in a Mixture of Enzyme Powder, Sodium Bicarbonate and Triacetin

The spray-dried enzyme powder prepared as described in Example 13 was evaluated for stability when stored for seven weeks at 40 ⁰ C as a mixture of the spray-dried powder in a mixture of triacetin and sodium bicarbonate. The spray-dried enzyme powder (0,556 g, 18.0 wt % protein) was added to 43.6 g of triacetin and 8.4 g of sodium bicarbonate (Grade 3DF (powder), Church & Dwight). The resulting mixture was stored at 40 ⁰ C, and a 2.63 g sample of the well-stirred mixture assayed in duplicate at 25 ⁰ C in a 100-mL reaction containing hydrogen peroxide (100 mM) and TURPINAL ^® SL (500 ppm), where the resulting concentrations of triacetin, sodium bicarbonate buffer (pH 7.2) and protein were 100 mM, 50 mM and 50 μg/mL, respectively. Comparison of the data in Table 10 with the data in Example 14, Table 9, demonstrates the improved stability of T. maritima perhydrolase/maltodextrin spray-dried enzyme powders when stored for five, six and seven weeks at 40 ⁰ C as a mixture with triacetin and solid sodium bicarbonate when compared to the stability of T. neapolitana perhydrolase/maltodextrin spray-dried enzyme powders when stored for five, six and seven weeks at 40 °C as a mixture with triacetin alone.

Table 10. Temperature stability of T. maritima perhydrolase/maltodextrin spray- dried enzyme powder during storage in a mixture of enzyme powder, sodium bicarbonate and triacetin at 40 ⁰ C. PAA (ppm) produced in 5 miπ at 25 ⁰ C by reaction of triacetin (100 mM) and H ₂ O ₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. maritima perhydrolase (50 μg protein/mL) and TURPiNAL ^® SL (500 ppm).

week 1 1138 week 2 1343 week 3 1242 week 4 1111 week 5 1149 week 6 1184 week 7 1109

EXAMPLE 16 Effect of Added Solvent on Peracetic Acid Production by Thermotoga neapolitana Perhydrolase

A first mixture of 90.0 g of deionized water, 0,350 g of TURPINAL ^® SL ((1-hydroxy-1-phosphonoethyl)phosphonic acid, 60 wt% in water; Thermphos International), and 3.20 g of 30 wt% hydrogen peroxide in water was adjusted to pH 7.2 with 50 % aqueous sodium hydroxide, and the final weight of the mixture adjusted to 100,0 g with deionized water. A second mixture of 55.76 g triacetin, 4.20 g of sodium bicarbonate, 2.50 g of CAB-O-SIL ^® M5 (Cabot), 0.270 g of spray-dried Thermotoga neapolitana perhydrolase (Example 10), and 37.43 g of one organic solvent selected from the group consisting of tripropylene glycol methyl ether (DOWANOL ^® TPM), dipropylene glycol methyl ether (DOWANOL ^® DPM), propylene glycol methyl ether (DOWANOL ^® PM), Diethyleπe glycol butyl ether (DOWANOL ^® DB), dipropylene glycol (DOWANOL ^® DPG), Methylene glycol, 1 ,2-propanediol, N-ethyl-2-ρyrroldinone, isopropanol, ethano!, ethyl lactate, or 1 ,3-propanediol was prepared. A 1.0 g aliquot of the second mixture was removed with rapid stirring (to suspend the undissolved solids) and mixed with 9.00 mL of the first mixture of hydrogen peroxide and TURPINAL ^® SL in water (pH 7.2) was added to with stirring at 25 ⁰ C; the resulting mixture contained 255 mM triacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL of spray-dried perhydrolase. A control reaction for each solvent was also run to determine the concentration of peracetic acid produced by chemicai perhydrolysis of triacetin by hydrogen peroxide in the absence of added protein.

Determination of the concentration of peracetic acid in the reaction mixtures was performed according to the method described by Karst et al. Aϋquots (0.040 ml_) of the reaction mixture were removed at predetermined times and mixed with 0.960 ml. of 5 mM phosphoric add in water; adjustment of the pH of the diluted sample to less than pH 4 immediately terminated the reaction. The resulting solution was filtered using an ULTRAFREE ^® MC-filter unit (30,000 Normal Molecular Weight Limit (NMWL) ₁ Millipore cat # UFC3LKT 00) by centrifugation for 2 min at 12,000 rpm. An aliquot (0.100 mL) of the resulting filtrate was transferred to 1.5-mL screw cap HPLC vial (Agilent Technologies, Palo Alto, CA; #5182-0715) containing 0.300 mL of deionized water, then 0.100 m L of 20 mM MTS (methyl-p-tolyl-suifide) in acetonitrile was added, the vials capped, and the contents briefly mixed prior to a 10 min incubation at ca. 25 ⁰ C in the absence of light. To each vial was then added 0.400 mL of acetonitrile and 0.100 mL of a solution of triphenyiphosphine (TPP ₁ 40 mM) in acetonitrile, the vials re-capped, and the resulting solution mixed and incubated at ca. 25 ⁰ C for 30 min in the absence of light. To each vial was then added 0.100 mL of 10 mM N.N-diethyl-m-toluamide (DEET; HPLC external standard) and the resulting solution analyzed by HPLC as described in Table 2.

HPLC Method:

SupeSco Discovery C8 column (10-cm X 4.0-mm, 5 μm) (cat. #569422-1 ⁾ ) w/precolumn Supelco Supelguard Discovery C8 (Sigma-Aldrich; cat # 59590- U); 10 microliter injection volume; gradient method with CH ₃ CN (Sigma-Aldrich; #270717) and deionized water at 1.0 mL/min and ambient temperature:

Time (min:sec) (% CH ₃ CN)

0:00 40

3:00 40

3:10 100

4:00 100 4:10 40 7:00 (stop) 40

The peracetic acid concentrations produced in 0.5 min, 1 min, 2 min, 5 min and 10 min for the reactions described above are listed in Table 11 , beiow.

TABLE 11

Dependence of peracetic acid (PAA) concentration on solvent addition using

255 mM triacetin, 254 mM hydrogen peroxide and 0.055 mg/mL of spray-dried

Thermotoga neapoiitana perhydrolase.

Enzyme PAA (ppm)

Solvent (μg/mL) 0.5 min 1 min 2 min 5 min 10 min

DOWANOL® PM 0 39 54 52 81 137

DOWANOL® DPM 0 136 41 106 386 ND

DOWANOL® TPM 0 23 25 11 1 93 180

DOWANOL® DB 0 107 102 105 157 218

DOWANOL® DPG 0 19 40 101 156 207

Triethyleπe glycol 0 36 53 110 76 307

1 ,2~propanediol 0 101 96 122 226 347

N-ethyl-2- pyrroldinone 0 37 49 60 77 133 isopropano! 0 70 13 147 150 242 ethaπol 0 68 33 150 356 479 ethyl lactate 0 88 91 98 121 137

1 ,3-propaπedioi 0 54 48 48 62 107

DOWANOL® PM 55 355 1327 1632 3156 5378

DOWANOL® DPM 55 846 972 1587 3209 4494

DOWANOL® TPM 55 439 539 1303 2710 3740

DOWANOL® DB 55 475 827 1719 3222 4863

DOWANOL® DPG 55 583 769 121 1 2784 4522

Triethylene glycol 55 325 834 1634 3229 51 16 1 ,2~propanedio! 55 507 903 1428 2921 4364

N-ethyi-2- pyrroldinoπe 55 243 837 1470 3033 4839 isopropanol 55 326 656 1175 2229 2860 ethaπol 55 408 584 1109 2235 2858 ethyl lactate 55 180 337 5736 1420 2554

1 ,3-propanediol 55 163 269 510 1086 1657

To demonstrate the stability of the spray-dried enzyme in a mixture of triacetin and an organic solvent, the mixtures of triacetin, sodium bicarbonate, CAB-O-SI L ^® M5 (Cabot), spray-dried Thermotoga neapolitana perhydrolase (Example 10), and either tripropylene glycol methyl ether (DOWANOL ^® TPM) or 1 ,2-propanedioI described above were stored for 24 h at ambient temperature, then a 1,0 g aliquot of each of these mixtures was removed with rapid stirring (to suspend the uπdissolved solids) and mixed with 9.0 mL of a freshly prepared (as described above) mixture of hydrogen peroxide and TURPINAL ^® SL in water (pH 7.2) with stirring at 25 ⁰ C; the resulting mixture contained 255 mM triacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL of spray-dried perhydroiase. A control reaction for each solvent was also run to determine the concentration of peracetic acid produced by chemical perhydrolysis of triacetin by hydrogen peroxide in the absence of added protein. Determination of the concentration of peracetic acid in the reaction mixtures was performed according to the method described by Karst et al. (Table 12).

TABLE 12 Stability of perhydrolase in triacetin solvent suspension, measured in reactions containing 255 mM triacetin and 254 mM hydrogen peroxide.

Enzyme PAA (ppm)

Solvent

(μg/mL) 0.5 min 1 min 2 miπ 5 miπ 10 miπ

DOWANOL® TPM 0 0 95 58 172 276

1 ,2-ρropanediol 0 16 38 35 171 397 DOWANOL® TPM 55 386 557 1078 2014 2717

1,2-propanedio! 55 566 768 1467 3093 4649

EXAMPLE 16 Comparison of Peracetic Acid Production by Thermotoga neapolitana

Perhydrolase in Presence or Absence of Added Solvent A first mixture of 40.0 g of deionized water, 0.1575 g of TURPINAL ^® SL

((i-hydroxy-i-phosphonoethyOphosphonic acid, 60 wt% in water; Thermphos International), and 1.44 g of 30 wt% hydrogen peroxide in water was adjusted to pH 7.2 with 50 % aqueous sodium hydroxide, and the final weight of the mixture adjusted to 46.87 g with deionized water, A second mixture of 2.78 g triacetin, 0.21 O g of sodium bicarbonate, 0.125 g of CAB-O -S I L ^® M 5 (Cabot) and 0.0135 g of spray-dried Thermotoga neapolitana perhydrolase (Example 10) was prepared, and the first mixture of hydrogen peroxide and TURPINAL ^® SL in water (pH 7.2) was added to the second mixture with stirring at 25 ⁰ C; the resulting mixture contained 255 mM triacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL of spray-dried perhydrolase. Determination of the concentration of peracetic acid in the reaction mixtures was performed according to the method described by Karst et ai, supra, A control reaction was also run to determine the concentration of peracetic acid produced by chemical perhydrolysis of triacetin by hydrogen peroxide in the absence of added perhydrolase.

The reaction described above was repeated, where 1 ,872 g of either propylene glycol monomethyl ether (DOWANOL ^® PM) or dipropyleneglycol monomethyl ether (DOWANOL ^® DPM), was substituted for an equivalent weight of water in the reaction mixture. A first mixture of 40.0 g of deionized water, 0.175 g of TURPINAL ^® SL, and 1.60 g of 30 wt % hydrogen peroxide in water was adjusted to pH 7.2 with 50 % aqueous sodium hydroxide, and the final weight of the mixture adjusted to 50.0 g with deionized water. A second mixture of 2.78 g triacetin, 1,872 g of either propylene glycol monomethyl ether (DOWANOL ^® PM) or dipropyleneglycol monomethyl ether (DOWANOL ^® DPM), 0.21O g of sodium bicarbonate, 0.125 g of CAB-O-S I L ^® M5 (Cabot) and 0.0135 g of spray-dried Thermotoga neapolitana perhydrolase (Example 10) was prepared, and 45.0 g of the first mixture of hydrogen peroxide and TURPINAL ^® SL in water (pH 7.2) was added to the second mixture with stirring at 25 ⁰ C; the resulting mixture (pH 6.5) contained 255 mM tπacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL of spray-dried perhydrolase. A control reaction was also run to determine the concentration of peracetic acid produced by chemical perhydrolysis of triacetin by hydrogen peroxide in the absence of added extract protein. The peracetic acid concentrations produced in 0.5 min, 1 min, 2 min, 5 min and 10 min for the three reactions described above are listed in Table 13, below.

TABLE 13

Dependence of peracetic acid (PAA) concentration on solvent addition using 255 mM triacetin, 254 mM hydrogen peroxide and 55 μg/mL of spray-dried Thermotoga neapolitana perhydrolase.

Enzyme PAA (ppm)

Solvent

(μg/mL) 0.5 min 1 min 2 min 5 min 10 min none 0 ND ND ND ND ND

DOWANOL® PM 0 89 90 205 318 498

DOWANOL® DPM 0 104 178 184 373 535

none 55 629 1359 2020 4274 6019

DOWANOL® PM 55 807 1390 2331 4439 5917

DOWANOL® DPM 55 787 1373 2566 5122 6528

EXAMPLE 17

Use of Solvent for in situ Peroxycarboxylic acid Generation Using a Two- Compartment

Spray-bottle Compared to Stirred Reactions

A first mixture of 100 g of 0.20 M sodium citrate buffer containing 2000 ppm TURPiNAL ^® SL ((1-hydroxy-1-phosphonoethy!) phosphonic acid, 60 wt% in water; Thermphos international), 280 g of deionized water, and 5.20 g of 30 wt % hydrogen peroxide in water was adjusted to pH 7.2 with 50 % aqueous sodium hydroxide, and the final weight of the mixture adjusted to 400 g with deionized water. A second mixture was separately prepared, containing 83.4 g of triacetin, 3.75 g of CAB-O-SI L ^® M5 (Cabot), 0.750 g of spray-dried Thermotoga neapotitana perhydrolase (Example 10), and 62.1 g of a single solvent selected from: propylene glycol methyl ether (DOWANOL ^® PM), tripropylene glycol methyl ether (DOWANOL ^® TPM), diethylene glycol methyl ether (DOWANOL ^® DM), propylene glycol n-butyl ether (DOWANOL ^® PNB), propylene glycol n-propyl ether (DOWANOL ^® PnP), propylene glycol monomethyl ether acetate (DOWANOL ^® PMA), dipropylene glycol, ethanoi, isopropanol, and 1 ,2-propanediol. In a first reaction at 25 ⁰ C, 1.0 g of the first mixture was stirred with 9.0 g of the second mixture for the first 30 - 60 seconds of the reaction (reaction pH of 6.5 - 6.0), and samples were withdrawn and analyzed for peracetic acid production; the resulting reaction mixture contained 255 mM triacetin, 103 mM hydrogen peroxide and 100 μg protein/mL of spray-dried perhydroiase. Determination of the concentration of peracetic acid in the reaction mixtures (TABLE 14, below) was performed according to the method described by Karst et a/., supra. A control reaction was also run to determine the concentration of peracetic acid produced by chemical perhydrolysis of triacetin by hydrogen peroxide in the absence of added perhydrolase.

The first mixture and second mixture prepared as described above were each separately charged to one of the two compartments of a two- compartment spray bottle (Custom Dual-Liquid Variable-Ratio Sprayer, Model DLS 200, manufactured by Take5 (Rogue River, OR)), where the bottle was set up to spray a mixture of 9 parts by weight of the first mixture with 1 part by weight of the second mixture. The two mixtures were sprayed into a 12.5 cm diameter crystallizing dish, and the resulting reaction mixture (reaction pH of 6.5 - 6.0) contained 255 mM triacetin, 100 mM hydrogen peroxide and 0.100 mg protein/mL of spray-dried perhydrolase. The sprayed reaction mixture was sampled at predetermined times and analyzed for peracetic acid (TABLE 14, below) according to the method described by Karst et al., supra. TABLE 14

Dependence of peracetic acid (PAA) concentration on solvent addition using

255 ΓTIM triacetin, 103 mM hydrogen peroxide and 100 μg/mL of spray-dried

Thermotoga neapolitana perhydrolase in stirred batch reactions and in a sprayed two-component mixture.

EXAMPLE 18 Peroxycarboxylic Acid Production Using Thermotoga maritima Perhydrolase

As An Enzyme Catalyst

5 Cloning and expression of perhydrolase from Thermotoga maritima is accomplished in accordance with the methods described in Examples 1-3 and 5, Fermentation of bacterial transformants expressing Thermotoga maritima perhydrolase is performed in accordance with preceding Example 6, and preparation of spray-dried Thermotoga maritima perhydrolase is accomplished 10 using methods described in Example 13. Additional information regarding techniques for cloning, expressing, and preparation of Thermotoga maritima perhydroiase is available in U.S. Ser. No. 12/143,375, filed June 20, 2008. A comparison of peracetic acid production by Thermotoga maritima perhydrolase in the presence and absence of added solvent is performed. A 15 first mixture of 40.0 g of deionized water, 0.1575 g of TURPINAL ^® SL ((1- hydroxy-1-phosphoπoethyl)phosphonic acid, 60 wt% in water; Therm phos International), and 1.44 g of 30 wt% hydrogen peroxide in water is adjusted to pH 7.2 with 50 % aqueous sodium hydroxide, and the final weight of the mixture adjusted to 46.87 g with deionized water. A second mixture of 2.78 g 20 triacetin, 0.21 O g of sodium bicarbonate, 0.125 g of CAB-O-SIL ^® M5 (Cabot) and 0.0135 g of spray-dried Thermotoga maritima perhydrolase is prepared, and the first mixture of hydrogen peroxide and TURPINAL ^® SL in water (pH 7.2) is added to the second mixture with stirring at 25 ⁰ C; the resulting mixture containing 255 mM triacetin, 254 mM hydrogen peroxide and 0.055 mg proteiπ/mL of spray-dried perhydrolase. Determination of the concentration of peracetic acid in the reaction mixtures is performed according to the method described by Karst et al., supra. A control reaction is also run to determine the concentration of peracetic acid produced by chemical perhydrolysis of triacetin by hydrogen peroxide in the absence of added perhydrolase.

The reaction described above is repeated, where 1.872 g of either propylene glycol monomethyl ether (DOWANOL ^® PM) or dipropyleneglycol monomethyl ether (DOWANOL ^® DPM), is substituted for an equivalent weight of water in the reaction mixture. A first mixture of 40.0 g of deionized water, 0.175 g of TURPINAL ^® SL, and 1.60 g of 30 wt % hydrogen peroxide in water is adjusted to pH 7.2 with 50 % aqueous sodium hydroxide, and the final weight of the mixture adjusted to 50.0 g with deionized water. A second mixture of 2.78 g triacetin, 1.872 g of either propylene glycol monomethyl ether (DOWANOL ^® PM) or dipropyleneglycol monomethyl ether (DOWANOL ^® DPM), 0.21O g of sodium bicarbonate, 0.125 g of CAB-O-SI L ^® M 5 (Cabot) and 0.0135 g of spray-dried Thermotoga maritima perhydrolase is prepared, and 45.0 g of the first mixture of hydrogen peroxide and TURPINAL ^® SL in water (pH 7.2) is added to the second mixture with stirring at 25 ⁰ C; the resulting mixture (pH 6.5) contained 255 mM triacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL of spray-dried perhydrolase. A control reaction is also run to determine the concentration of peracetic acid produced by chemicai perhydrolysis of triacetin by hydrogen peroxide in the absence of added extract protein. The peracetic acid concentrations produced in 0.5 min, 1 min, 2 min, 5 min and 10 min for the three reactions described above are measured and recorded.

EXAMPLE 19

Use of Solvent For In Situ Peroxycarboxylic acid Generation Using Two- Compartment Spray Device Compared to Stirred Reaction and Using Thermotoga maritima Perhvdrolase A first mixture of 100 g of 0.20 M sodium citrate buffer containing 2000 ppm TURPINAL ^® SL ((i-hydroxy-i-phosphonoethyOphosphonic acid, 60 wt% in water; Thermphos International), 280 g of deionized water, and 5.20 g of 30 wt % hydrogen peroxide in water is adjusted to pH 7.2 with 50 % aqueous sodium hydroxide, and the final weight of the mixture adjusted to 400 g with deionized water. A second mixture is separately prepared, containing 83.4 g of triaoetin, 3.75 g of CAB-O-SIL ^® M5 (Cabot), 0.750 g of spray-dried Thermotoga maritima perhydrolase (Example 13), and 62.1 g of a single solvent selected from: propylene glycol methyl ether (DOWANOL® PM), tripropylene glycol methyl ether (DOWANOL ^® TPM), diethyleπe glycol methyl ether (DOWANOL® DM), propylene glycol n-butyl ether (DOWANOL® PNB), propylene glycol n- propyl ether (DOWANOL ^® PnP), propylene glycol monomethyl ether acetate (DOWANOL ^® PMA), dipropylene glycol, ethaπol, isopropanol, and 1,2- propanediol. In a first reaction at 25 ⁰ C, 1 ,0 g of the first mixture is stirred with 9.O g of the second mixture for the first 30 - 60 seconds of the reaction (reaction pH of 6.5 - 6.0), and samples are withdrawn and analyzed for peracetic acid production; the resulting reaction mixture containing 255 mM triacetin, 103 mM hydrogen peroxide and 100 μg protein/mL of spray-dried perhydrolase. Determination of the concentration of peracetic acid in the reaction mixtures is performed according to the method described by Karst et a/., supra. A control reaction is also run to determine the concentration of peracetic acid produced by chemical perhydrolysis of triacetin by hydrogen peroxide in the absence of added perhydrolase.

The first mixture and second mixture prepared as described above are each separately charged to one of the two compartments of a two- compartment spray bottle (Custom Dual-Liquid Variable-Ratio Sprayer, Model DLS 200, manufactured by Take5 (Rogue River, OR)), where the bottle is set up to spray a mixture of 9 parts by weight of the first mixture with 1 part by weight of the second mixture. The two mixtures are sprayed into a 12.5 cm diameter crystallizing dish, and the resulting reaction mixture (reaction pH of 6.5 - 6.0) containing 255 mM triacetin, 100 mM hydrogen peroxide and 0.100 mg protein/mL of spray-dried perhydrolase. The sprayed reaction mixture is sampled at predetermined times and analyzed for peracetic acid according to the method described by Karst et a/., supra.

EXAMPLE 20 Exemplary Two-Component System

One example of a two-component in situ peroxycarboxylic acid disinfectant formulation is described below.

Component A mol/L grams triacetin 0.100 21.82

T. neapolitana perhydrolase/excipient 0.20 sodium bicarbonate 0.050 4.20

Component B

H ₂ O ₂ (30 wt %): 0.100 11.33

TURPiNAL ^® SL {60 wt%, 0.1 % final)

1.67 water (deionized) 960.78

Total weight (grams) 1000.00

For the two-component in situ peroxycarboxylic acid disinfectant formulation described above, Component A comprises ca. 2.6 wt % of the combined weight of Components A and B ₁ and the weight ratio of Component B to Component A is ca. 38:1. in certain applications for a two-component in- situ peroxycarboxylic acid disinfectant formulation, it may be desirable for the ratio of Component B to Component A to be within a range of from 1:1 to 10:1 , where from 10 parts to 1 part (by weight) of Component B is mixed with one part (by weight) of Component A to produce a peroxycarboxylic acid at a concentration efficacious for disinfection. For example, in a first application a two-compartment spray bottle such as a dual-liquid fixed ratio sprayer (Model DLS100, Takeδ) or a dual-liquid variable ratio sprayer (Model DLS200, Take5) is utilized, where a maximum ratio of Component B to Component A of 10:1 is employed, in a second application, a single bottle containing two separate compartments separated by a breakable seal is employed, where the ratio of the volume of the two separate compartments is 1 : 1 , or 5: 1 or 10: 1. In each of these applications, the two-component formulation cannot be mixed at the desired ratio of Component A to Component B to provide the desired concentration of reactaπts and final concentration of products.

EXAMPLE 21 Perhydrolase Activity Assay of Thermotoqa neapolitana Acetyl Xylan Esterase Variants

Libraries of Thermotoga neapolitana mutants were prepared as described in Example 4 of co-owned, co-fiied, and copending U.S. Patent Application having attorney docket number CL4392 US NA entitled "Improved Perhydrolases for Enzymatic Peracid Production". Briefly, saturation mutagenesis was conducted at amino acid residues F213, I276, C277, and N93 of SEQ ID NO: 6 so that each of the other 19 possible amino acids were individually introduced to each specified position.

The mutations were made using QUIKCHANGE ^® (Stratagene, La JoIIa, CA) kit according to the manufacturer's instructions. Amplified plasmids were treated with 1 U of Dpn\ at 37 ⁰ C for 1 hour. Treated plasmids were used to transform chemically competent E. coli XL1-Biue (Stratagene) (residues 213, 276 and 277) or chemically competent E. co// TOP10F ¹ (Invitrogen, Carlsbad, CA) (residue 93). Transformants were plated on LB-agar supplemented with 0.1 mg ampicillin/mL and grown overnight at 37 ⁰ C. Up to five individual colonies were picked and the plasmid DNA sequenced to confirm the expected mutations.

Individual colonies of mutants were picked into 96-well plates containing 0.1 mL LB with 0.1 mg ampiciiiin/mL, and grown overnight at 37 ⁰ C without shaking. 0.003 mL of overnight culture was transferred to an "induction plate" (96 deep-wel!) containing 0.3 mL LB, 0.5 mM IPTG and 0.1 mg ampicillin/mL. Induction plates were grown overnight at 37 °C with shaking. 0.01 mL of Induction culture was transferred to "Lysis plate" (96-well) containing 0.09 mL of 56 mg/mL CELYTIC ™ Express (Sigma Aldrich, St. Louis, MO). Plates were slightly agitated first, before incubating at 25 ^D C for 30 minutes. Approximately 0.01 mL of Lysis culture was transferred to "Assay plate" (96-well) containing 0.09 mL "Assay solution pH 5.0" (100 mM triacetin, 100 mM hydrogen peroxide, 50 mM acetic acid pH 5.0). Approximately 0.01 mL of Lysis culture was also transferred to "Assay plate pH 7.5" (96-we!l) containing 0.09 mL "Assay solution pH 7,5" (100 mM triacetin, 100 mM hydrogen peroxide, 50 mM sodium phosphate pH 7,5). Plates were gently agitated for 30 seconds before incubating at ambient temperature for 10 minutes. The assay was quenched by addition of 0.1 mL of "Stop buffer" (100 mM ortho-phenylenediamine (OPD), 500 mM NaH ₂ PO ₄ pH 2.0). Plates were gently agitated for 30 seconds before incubating at 25° C for 30 minutes. The absorbance was read at 458 nm without a lid using a SPECTRAMAX ^® Plus384 plus (Molecular Devices, Sunnyvale, CA). Analysis of the results indicated four variants that demonstrated significantly greater perhydroiase activity compared to the native enzyme (Tables 15 and 16). All four are changes of the cysteine at residue 277 (C277A, C277V, C277S, and C277T; see SEQ ID NO: 19).

Table 15. Perhydroiase activity (U/mL) at pH 5.0 of T, neapolitana acetyl xyiaπ esterase variants.

Table 16. Perhydrolase activity at pH 7.5 of T. neapolitana acetyl xylan esterase variants.

EXAMPLE 22 Expression of Thermotoga neapolitana Acetyl Xylan Esterase Variants in E.

CO// KLP 18

Plasmids with confirmed acetyl xylan esterase mutations were used to transform E. coli KLP18 (Example 3). Transformants were plated onto LB- ampϊcilltn (100 μg/mL) plates and incubated overnight at 37 ⁰ C, Cells were harvested from a ptate using 2.5 ml_ LB media suppiemented with 20 % (v/v) glycerol, and 1.0 mL aliquots of the resulting cell suspension frozen at - 80 ⁰ C. One mL of the thawed cell suspension was transferred to a 1-L APPLIKON ^® Bioreactor (Applikon ^® Biotechnology, Foster City, CA) with 0.7 L medium containing KH ₂ PO ₄ (5.0 g/L), FeSO ₄ heptahydrate (0.05 g/L), MgSO ₄ heptahydrate (1.0 g/L), sodium citrate dihydrate (1.90 g/L), yeast extract (Amberex 695, 5.0 g/L), Biospumex153K antifoam (0.25 mL/L, Cognis Corporation), NaCI (1.0 g/L), CaCI ₂ dihydrate (0.1 g/L), and NIT trace elements solution (10 mL/L). The trace elements solution contained citric acid monohydrate (10 g/L), MnSO ₄ hydrate (2 g/L), NaC! (2 g/L), FeSO ₄ heptahydrate (0.5 g/L), ZnSO ₄ heptahydrate (0.2 g/L), CuSO ₄ pentahydrate (0.02 g/L) and NaMoO ₄ dihydrate (0.02 g/L). Post sterilization additions included glucose solution (50% w/w, 6.5 g) and ampicϋlin (25 mg/mL) stock solution (2.8 mL). Glucose solution (50% w/w) was also used for fed batch. Glucose feed was initiated 40 min after glucose concentration decreased below 0.5 g/L, starting at 0.03 g feed/min and increasing progressively each hour to 0.04, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.12, and 0.14 g/min respectively; the rate remaining constant afterwards. Giucose concentration in the medium was monitored, and if the concentration exceeded 0.1 g/L the feed rate was decreased or stopped temporarily. Induction was initiated at OD ₅₅₀ = 50 with addition of 0.8 mL IPTG (0.05 M). The dissolved oxygen (DO) concentration was controlled at 25% of air saturation, first by agitation (400- 1000 rpm), and following by aeration (0,5-2 slpm). The temperature was controlled at 37 °C ₍ and the pH was controlled at 6.8; NH ₄ OH (29% w/w) and H ₂ SO ₄ (20% w/v) were used for pH control. The cells were harvested by centrifugation (5,000 x g for 15 minutes) at 20 h post IPTG addition..

EXAMPLE 23 Preparation of Cell Lysates Containing Semi-Purified T. neapolitana Acetyl Xylan Esterase or T. neapoHtana Variant Acetyl Xylan Esterases A cell culture of E. coli KLP18/pSW196 (Thermotoga neapolitana wild- type perhydrolase) was grown as described in Example 6. The resulting cell paste was resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0 supplemented with 1.0 mM DTT. Resuspended cells were passed through a French pressure ceil twice to ensure >95% cell lysis. Lysed cells were centrifuged for 30 minutes at 12,000 x g, and the supernatant was heated at 75 ⁰ C for 20 minutes, followed by quenching in an ice bath for 2 minutes.

Precipitated protein was removed by centrifugation for 10 minutes at 11 ,000 x g, SDS-PAGE indicated that the CE-7 enzyme comprised approximately 85-90 % of the total protein in the heat-treated extract supernatant.

Cell cultures of E. coli KLP18/pSW196/C277S {Thermotoga neapolitana C277S variant perhydrolase), E. coli KLP18/pSW196/C277V {Thermotoga neapolitana C277V variant perhydrolase), E. coli KLP18/pSW196/C277A (Thermotoga neapolitana C277A variant perhydrolase), and E. coli KLP18/pSW196/C277T (Thermotoga neapolitana C277T variant perhydrolase) were each grown as described in Example 22. The resulting cell pastes were resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0 supplemented with 1.0 mM DTT. Resuspended ceils were passed through a French pressure cell twice to ensure >95% cell lysis. Lysed cells were centrifuged for 30 minutes at 12,000 x g, and the supernatant was heated at 75 ⁰ C for 20 minutes, followed by quenching in an ice bath for 2 minutes. Precipitated protein was removed by centrifugation for 10 minutes at 11 ,000 x g. SDS-PAGE indicated that the CE-7 enzyme comprised approximately 85-90 % of the total protein in the heat- treated extract supernatant.

EXAMPLE 24 Specific Activity and Perhydrolysis/Hydrolysis ratio of T neapolitana Acetyl

Xylan Wild-type Esterase and C277 Esterase Variantss Reactions (40 mL total volume) were run at 25 °C in phosphate buffer (50 mM, pH 7.2) containing triacetin (100 mM), hydrogen peroxide (100 mM) and one of the following acetyl xylan esterase mutants: T. neapolitana C277S variant perhydrolase (0.010 mg/mL of heat-treated extract total protein from E. coli KLP18/pSW196/C277S), T. neapolitana C277T variant perhydrolase (0.010 mg/mL of heat-treated extract tota! protein from E. coli KLP18/ pSW196/C277T), T. neapolitana C277A variantperhydrolase (0.0125 mg/mL of heat-treated extract total protein from E coli KLP18/pSW196/C277A), and T. neapoHtana C277V variantperhydrotase (0.0125 mg/mL of heat-treated extract total protein from E coli KLP18/pSW196/C277V) (prepared as described in Example 23). Reactions were stirred for only the first 30 seconds of reaction to initially mix the reactants and enzyme.

A reaction was also run under indentical conditions to that described immediately above using 0.050 mg/mL of heat-treated extract total protein isolated from E coli KLP18/pSW196 (expressing Thermotoga neapoHtana wild- type acetyl xylan esterase (Example 1)), where the heat-treated extract supernatant was prepared according to the procedure of Example 23.

Two samples from each of the reaction mixtures described above were simultaneously withdrawn after the first minute of each reaction, and every two minutes thereafter for fifteen minutes, where one of the two samples was analyzed for peracetic acid, and the second sample was analyzed for total acetic acid produced from both enzymatic hydrolysis of triacetin and from subsequent conversion of peracetic acid in sample to acetic acid by reaction with methyl-p-tolyl sulfide (MTS, see below).

Measurement of the rate of peracetic acid production in the reaction mixture was performed using a modification of the method described by Karst et a/., supra. A sample (0.040 mL) of the reaction mixture was removed at a predetermined time and immediately mixed with 0.960 mL of 5 mM phosphoric acid in water to terminate the reaction by adjusting the pH of the diluted sample to less than pH 4. The resulting solution was filtered using an ULTRAFREE ^® MC-fϋter unit (30,000 Normal Molecular Weight Limit (NMWL), Millipore Corp., Bilterica, MA; cat # UFC3LKT 00) by centrifugation for 2 min at 12,000 rpm. An aliquot (0.100 mL) of the resulting filtrate was transferred to a 1.5-mL screw cap HPLC vial (Agilent Technologies, Palo Aito, CA; #5182-0715) containing 0.300 mL of deionized water, then 0.100 mL of 20 mM MTS (methyl-p-to!yl sulfide) in acetonithfe was added, the vial capped, and the contents briefly mixed prior to a 10 mm incubation at ca. 25 ⁰ C in the absence of light. To the vial was then added 0.400 mL of acetonitrile and 0.100 mL of a solution of triphenylphosphine (TPP, 40 mM) in acetonitrile, the vial re-capped, and the resulting solution mixed and incubated at ca. 25 ⁰ C for 30 min in the absence of light. To the vial was then added 0.100 mL of 10 mM N.N-diethyl-m- toluamide (DEET; HPLC external standard) and the resulting solution analyzed by HPLC for MTSO (methyl-p-tolyl sulfoxide), the stoichiometric oxidation product produced by reaction of MTS with peracetic acid. A control reaction was run in the absence of added extract protein or triacetin to determine the rate of oxidation of MTS in the assay mixture by hydrogen peroxide, for correction of the rate of peracetic acid production for background MTS oxidation. HPLC method: Supelco Discovery C8 column (10-cm X 4.0-mm, 5 μm) (catalog #569422-U) with Supelco Supeiguard Discovery C8 precolumπ (Sigma-Aldrich; catalog # 59590-U); 10 microliter injection volume; gradient method with CH ₃ CN (Sigma-Aldrich; catalog #270717) and deionized water at 1.0 mL/min and ambient temperature (Table 17).

Table 17. HPLC Gradient for analysis of peracetic acid.

For determination of the rate of perhydrolase-catalyzed acetic acid production in the reaction, a sample (0.900 mL) of the reaction mixture was removed at a predetermined time and immediately added to a 1.5 mL- microcentrifuge tube containing 0.040 mL of 0.75 M H ₃ PO ₄ , and the resulting solution briefly mixed to terminate the reaction at pH 3.0 - 4,0. To the tube was then added 0.020 mL of a solution of 10 mg/mL of Aspergillus niger catalase (Sigma-Aldrich; C3515) in 50 mM phosphate buffer pH (7.2), and the resulting solution mixed and allowed to react for 15 minutes at ambient temperature to disproportionate uπreacted hydrogen peroxide to water and oxygen. To the tube was then added 0.040 mL of 0.75 M H ₃ PO ₄ and the resulting solution mixed and filtered using an ULTRAFREE ^® MC-filter unit (30,000 Normal Molecular Weight Limit (NMWL) ₅ Millipore Corp., cat # UFC3LKT 00) by centrifugation for 2 min at 12,000 rpm. An aliquot (0.100 mL) of the resulting filtrate was mixed with 0.150 mL of 20 mM MTS (methyl-p-tolyl sulfide) in acetonitrile, and the resulting solution was incubated for 10 min at ca. 25 ⁰ C in the absence of Sight. The concentration of acetic acid in the sample produced by both enzymatic hydrolysis of triacetin and conversion of peracetic acid to acetic acid by reaction with MTS was determined using a gas chromatograph (GC) equipped with a flame ionization detector (FID) and a DB-FFAP column (length, 15 m; ID, 0.530 mm; film thickness, 1.00 μm); a fresh injection port liner was employed for each rate determination (total of eight sample analyses) to avoid build up of phosphoric acid in the injection port liner over time.

The Thermotoga neapolitana acetyl xylan esterase variants had a significantly-higher specific activity for perhydrolysis of triacetin than the wild- type esterase (Table 18). The perhydrolysis/hydrolysis ratios for the T. neapolitana acetyl xyian esterase variants were determined by dividing the rate of PAA production (perhydrolysis rate) by the rate of hydrolysis of triacetin to acetic acid (hydrolysis rate) (calculated from the rate of total acetic acid production in the assay method from both PAA and acetic acid, and corrected for the rate of peracetic acid production); the P/H ratio of the T. neapolitana acetyl xylan esterase variants were ca. equal to or greater than the P/H ratio for the T. neapolitana wild-type acetyl xylan esterase (Table 18).

Table 18.

Thermotoga enzyme perhydrolysis hydrolysis P/H specific neapolitana concen. rate rate ratio activity perhydrolase (μg/mL) (mM/min) (mM/min) (U/mg protein) wild type 50 3.61 1.22 3.0 72

C277S 10 4.40 1.61 2.7 440

C277T 10 4.24 0.81 5.2 424

C277A 12.5 4.14 1.43 2.9 331

C277V 12.5 3.70 0.88 4.2 296 EXAMPLE 25

Cioninq and Expression of Acetyl Xylan Esterase from Thermotoga maritima A gene encoding acetyl xylan esterase from T. maritima as reported in GENBANK ^® (accession # NP_227893.1 ) was synthesized (DNA 2.0, Menlo Park CA). The gene was subsequently amplified by PCR (0.5 min at 94 ⁰ C, 0.5 min at 55 ⁰ C, 1 min at 70 ⁰ C, 30 cycles) using primers identified as SEQ ID NO: 52 and SEQ ID NO: 53. The resulting nucleic acid product (SEQ ID NO: 54) was cut with restriction enzymes Pstl and Xbal and subcioned between the Pst\ and Xba\ sites in pUC19 to generate the plasmid identified as pSW207. A gene encoding an acetyl xylan esterase from T. maritima MSB8 as reported in GENBANK ^® (Accession no. NP_227893.1 ) was synthesized using codons optimized for expression in E. coli (DNA 2.0, Menlo Park CA). The gene was subsequently amplified by PCR (0.5 min at 94 ⁰ C, 0.5 min at 55 ⁰ C, 1 min at 70 ⁰ C, 30 cycles) using primers identified as SEQ ID NO:55 and SEQ ID NO:56. The resulting nucleic acid product was cut with restriction enzymes EcoR\ and Pst\ and subcioned between the EcoR\ and Psrl sites in pTrc99A (GENBANK ^® Accession no. M22744) to generate the plasmid identified as pSW228 (containing the codon-optimized T. maritima coding sequence SEQ ID NO: 57). The plasmids pSW207 and pSW228 were used to transform E. coli KLP18 (U.S. Patent Application Pub. No. 2008/0176299) to generate the strain identified as KLP18/pSW207 and KLP18/pSW228, respectively. KLP18/pSW207 and KLP18/pSW228 were gown in LB media at 37 ^D C with shaking up to OD ₆ oonm = 0.4-0.5, at which time IPTG was added to a final concentration of 1 mM, and incubation continued for 2-3 h. Cells were harvested by centπfugatϊon and SDS-PAGE was performed to confirm expression of the perhydrolase at 20-40% of total soluble protein.

EXAMPLE 26

Construction of ThermotOQa maritima Acetyl Xylan Esterase Variants at Residue C277

The C277 (Cys277) position of T. maritima acetyl xylan esterase was changed to each of VaI ₁ Ala, Ser and Thr using oligonucleotide primer pairs (Table 19) that were designed based on the codon optimized sequence of T. maritima acetyl xylan esterase (SEQ ID NO:57) in the plasmid pSW228. The mutations were made using QUIKCHANGE ^® (Stratagene) according to the manufacturer's instructions. Amplified plasmids were treated with 1 U of Dpnl at 37 ⁰ C for 1 hour. Treated plasmids were used to transform chemically competent E. co// XL1-Blue (Stratagene). Transformants were plated on LB- agar supplemented with 0.1 mg ampicϋlin/mL and grown overnight at 37 ⁰ C. Up to five individual colonies were picked and the plasmid DNA sequenced to confirm the expected mutations.

Table 19. Oligonucleotides used to change residue 277 in T. maritima. forward 5' to 3' reverse 5 ¹ to 3'

Tma_ C277Vf Tma_C277Vr (SEQ ID NO:58) ggacaacatcG TG cctccttcta (SEQ ID NO:59) TAGAAGGAGGCACGATGTTGTCC Tma_C277Af Tma_C277Ar (SEQ ID NO:60) ggacaacatcGCGcctccttcta (SEQ ID NO:61 ) TAGAAGGAGGCGCGATGTTGTCC Tma_C277Sf Tma_C277Sr (SEQ iD NO:62) gga caa catcTCAcctccttcta (SEQ ID NO:63) TAGAAGGAGGTGAGATGTTGTCC Tma_C277Tf Tma_C277Tr (SEQ ID NO:64) gg acaacatcAC Ccctccttcta (SEQ ID NO-65) TAGAAG G AG GG GTG ATGTTGTCC

EXAMPLE 27 Expression of Thermotoga maritima Acetyl Xylan Esterase Variants in

E, coli KLP 18 Plasmids with confirmed acetyl xylan esterase mutations were used to transform £ coli KLP18 (Example 3). Transformants were grown in LB media at 37 °C with shaking up to OD ₆ oonm = 0.4-0.5, at which time IPTG was added to a final concentration of 1 mM, and incubation continued for 2-3 h. Cells were harvested by centrifugation and SDS-PAGE was performed to confirm expression of the acetyl xylan esterase at 20-40% of total soluble protein. EXAMPLE 28 Preparation of CeIi Lysates Containing Semi-Purified T. maritime Acetyl Xylan

Esterase Mutants

CeIi cultures (prepared as described in Example 27) were grown using a fermentation protocoi similar to that described in Example 7 at a 1-L scale (Applikon). Cells were harvested by centrifugation at 5,000 x g for 15 minutes then resuspended (20% w/v) in 50 mM phosphate buffer pH 7,0 supplemented with 1.0 mM DTT. Resuspended cells were passed through a French pressure cell twice to ensure >95% cell lysis. Lysed cells were centrifuged for 30 minutes at 12,000 x g, and the supernatant was heated at 75° C for 20 minutes, followed by quenching in an ice bath for 2 minutes. Precipitated protein was removed by centrifugation for 10 minutes at 11 ,000 x g. SDS- PAGE indicated that the CE-7 enzyme comprised approximately 85-90% of the total protein in the preparation.

EXAMPLE 28 Specific Activity and Perhydrolysis/Hydrolysis ratio of T. maritime Acetyl

Xylan Wild-type Esterase and C277 Esterase Variants Reactions (40 mL total volume) were run at 25 ⁰ C in phosphate buffer (50 mM, pH 7.2) containing triacetin (100 mM), hydrogen peroxide (100 mM) and one of the following acetyl xylaπ esterase variants: T. maritima C277S variant perhydrolase (0.010 mg/mL of heat-treated extract total protein from E. coli KLP18/pSW228/C277S), T. maritima C277T variantperhydrolase (0.010 mg/mL of heat-treated extract total protein from E. coli KLP18/ pSW228/C277T), T. maritima C277A variant perhydrolase (0.0125 mg/mL of heat-treated extract total protein from E. coii KLP18/pSW228/C277A), and T. maritima C277V variant perhydrolase (0.0125 mg/mL of heat-treated extract total protein from E. coli KLP18/pSW228/C277V) (prepared as described in Example 27). Reactions were stirred for only the first 30 seconds of reaction to initially mix the reactants and enzyme.

A reaction was also run under indentical conditions to that described immediately above using 0.050 mg/mL of heat-treated extract total protein isolated from E. coli KLP18/pSW228 (expressing Thermotoga maritima wild- type acetyl xylan esterase (Example 25)), where the heat-treated extract supernatant was prepared according to the procedure of Example 27.

Two samples from each of the reaction mixtures described above were simultaneously withdrawn after the first minute of each reaction, and every two minutes thereafter for fifteen minutes, where one of the two samples was analyzed for peracetic acid using a modification of the method described by Karst et a/., supra, and the second sample was analyzed for total acetic acid produced from both enzymatic hydrolysis of triacetin and from subsequent conversion of peracetic acid in sample to acetic acid by reaction with methyl-p- tolyl sulfide (MTS) (see Example 8).

The Thermotoga maritima acetyl xylan esterase mutants had a significantly-higher specific activity for perhydroiysis of triacetin than the wild- type esterase (Table 20). The perhydrolysis/hydrolysis ratios for the T. maritima acetyl xylan esterase variants were determined by dividing the rate of PAA production (perhydroiysis rate) by the rate of hydrolysis of triacetin to acetic acid (hydrolysis rate) (calculated from the rate of total acetic acid production in the assay method from both PAA and acetic acid, and corrected for the rate of peracetic acid production); the P/H ratio of the T ^" . maritima acetyl xylan esterase variants were ca. equal to or greater than the P/H ratio for the T. neapolitana wild-type acetyl xylan esterase (Table 20).

Table 20.

Thermotoga enzyme perhydroiysis hydrolysis P/H specific maritima concen. rate rate ratio activity perhydrolase (μg/mL) (m IWm in) (mM/min) (U/mg protein) wild type 50 3.06 0.47 6.5 61

C277S 10 7.77 0.48 16 111

C277T 10 6.93 1.05 6.6 693

C277A 10 4.27 0.088 48 427

C277V 10 4.25 0.062 68 425

EXAMPLE 29

Peracetic Acid Production Usinq Perhydrolases Reactions (100 mL total volume) containing triacetin (2 mM), hydrogen peroxide (10 mM) and from 0.1 μg/mL to 2.0 μg/mL heat-treated cell extract protein (prepared as described above, where the heat-treatment was performed at 85 ^β C for 20 min) were run in 10 mM sodium bicarbonate buffer (initial pH 8.1) at 20 ⁰ C. Determination of the concentration of peracetic acid in the reaction mixtures was performed according to the method described by Karst et a/., supra. The peracetic acid concentrations produced in 1 min, 5 min, 20 min, 40 min and 60 min are listed in Table 21.

Table 21 : Dependence of peracetic acid (PAA) concentration on perhydrolase concentration in reactions containing triacetin (2 mM) and hydrogen peroxide (10 mM) in sodium bicarbonate buffer (10 mM, initial pH 8.1) at 20 ⁰ C, using heat-treated extract protein from E. coli KLP18/pSW228 (Thermotoga maritime wild-type perhydrolase) or E. coli KLP18/pSW228/C277S {Thermotoga maritima C277S variant perhydrolase) (duplicate reactions).

Thermotoga enzyme PAA, PAA, PAA ₁ PAA, PAX ^" maritima triacetin concen. 1 min 5 min 20 min 40 min 60 min perhydroiase (mM) (μg/mL) (ppm) (ppm) (ppm) (ppm) (ppm) no enzyme 2 0 0 0 1 1 3

wild type 2 0.2 0 2 7 13 19 wild type 2 0.2 0 1 5 11 15 wild type 2 0.5 0 2 12 19 25 wild type 2 0.5 0 2 12 21 26 wild type 2 1.0 0 5 20 29 31 wild type 2 1.0 0 5 19 30 31 wild type 2 2.0 1 1 1 24 24 20 wild type 2 2.0 1 1 1 29 29 21

C277S 2 0.2 0 4 18 18 18

C277S 2 0.2 0 4 18 17 18

C277S 2 0.5 1 12 39 54 64 C277S 2 0.5 1 10 34 52 64

C277S 2 1.0 18 26 59 69 63

C277S 2 1.0 18 25 60 70 64

C277S 2 2.0 9 38 66 60 48

C277S 2 2.0 9 34 69 61 49

EXAMPLE 30

Peracetic Acid Production Usiπq Perhydrolases

Reactions (10O mL total volume) containing triacetin (20 mM), hydrogen peroxide (10 mM) and from 0.1 μg/mL to 2.0 μg/mL heat-treated cell extract protein (prepared as described above, where the heat-treatment was performed at 85 ⁰ C for 20 min) were run in 10 mM sodium bicarbonate buffer (initial pH 8.1 ) at 20 ⁰ C. Determination of the concentration of peracetic acid in the reaction mixtures was performed according to the method described by Karst et a/., supra. The peracetic acid concentrations produced in 1 min, 5 min, 20 min, 40 min and 60 min are listed in Table 22.

Table 22: Dependence of peracetic acid (PAA) concentration on perhydrolase concentration in reactions containing triacetin (20 mM) and hydrogen peroxide (10 mM) in sodium bicarbonate buffer (10 mM, initial pH 8.1 ) at 20 ⁰ C, using heat-treated extract protein from E. coli KLP18/pSW228 (Thermotoga maritima wild-type perhydrolase) or E coli KLP18/pSW228/C277S {Thermotoga maritima C277S variant perhydrolase) (duplicate reactions).

Thermotoga enzyme PAA PAA, PAA PAA^ PAA ₁ maritima triacetin concen. 1 min 5 min 20 min 40 min 60 min perhydrolase (mM) (μg/mL) (ppm) (ppm) (ppm) (ppm) (ppm) no enzyme 20 0 2 3 3 7 9

wild-type 20 0.2 3 10 15 27 35 wild-type 20 0.2 4 9 19 32 41 wild-type 20 0.5 3 9 21 39 52 wild-type 20 0.5 3 8 22 39 54 wϋd-type 20 1.0 4 13 35 62 82 wild-type 20 1.0 4 12 37 67 wild-type 20 2.0 9 20 52 91 122 wild-type 20 2.0 10 20 52 87 1 14

C277S 20 0.2 7 16 67 109 148

C277S 20 0.2 9 24 67 112 144

C277S 20 0.5 16 43 140 202 260

C277S 20 0.5 17 48 148 228 272

C277S 20 1.0 24 75 230 289 353

C277S 20 1.0 26 97 232 297 372

C277S 20 2.0 32 130 318 402 443

C277S 20 2.0 37 135 323 401 430

EXAMPLE 31

Peracetic Acid Production Usinq Perhydrolases

Reactions (40 mL total volume) were run at 25 ⁰ C in phosphate buffer (50 mM, pH 7.2) containing triacetin (100 mM), hydrogen peroxide (100 mM) and from 10 μg/mL to 50 μg/mL of heat-treated T. neapolitana or T. maritima wild-type or C277 variant perhydrolases (as heat-treated cell extract protein prepared as described above, where the heat-treatment was performed at 75 ⁰ C for 20 min). Reactions were stirred for only the first 30 seconds of reaction to initially mix the reactants and enzyme. Determination of the concentration of peracetic acid in the reaction mixtures was performed according to the method described by Karst et a/., supra. The peracetic acid concentrations produced in 1 min, 5 min, and 30 min are listed in Table 23.

Table 23: Peracetic acid (PAA) production in reactions containing triacetin (100 mM) and hydrogen peroxide (100 mM) in phosphate buffer (50 mM, pH 7.2) at 25 ⁰ C, using heat-treated T. neapolitana or T. maritima wild-type or C277 mutant perhydrolases.

perhydrolase triacetin H ₂ O ₂ enzyme PAA, PAA, PAA, (mM) (mM) concen. 1 min 5 min 30 min

(μg/mL) (ppm) (ppm) (ppm) no enzyme 100 100 0 63 54 80

T. maritima wild-type 100 100 50 529 1790 3785

T. maritima C277S 100 100 10 979 3241 4635

T. maritima C277T 100 100 10 933 2882 3527

T. maritima C277A 100 100 10 442 2018 2485

T, maritima C277V 100 100 10 577 1931 2278

T. neapolitana wild-type 100 100 50 514 1837 3850

T. neapoiitana C277S 100 100 10 606 2237 4609

T, neapolitana C277T 100 100 10 634 2198 3918

T, neapolitana C277A 100 100 12.5 516 2041 3735

T. neapolitana C277V 100 100 12.5 451 1813 2758

EXAMPLE 32

Peracetic Acid Production Usinq Perhydrolases

Reactions (10 mL total volume) were run at 25 ⁰ C in sodium bicarbonate buffer (1 mM, initial pH 6.0) containing triacetin (100 mM or 150 mM), hydrogen peroxide (100 mM, 250 mM or 420 mM) and heat-treated T. neapolitana or T. maritima wild-type, C277S or C277T varant perhydrolases (as heat-treated cell extract protein prepared as described above, where the heat-treatment was performed at 75 ⁰ C for 20 min; concentrations as listed in Table 24). Reactions run using 420 mM hydrogen peroxide additionally contained 500 ppm TURPINAL ^® SL Reactions were stirred for only the first 30 seconds of reaction to initially mix the reactants and enzyme. Determination of the concentration of peracetic acid in the reaction mixtures was performed according to the method described by Karst et a/., supra. The peracetic acid concentrations produced in 1 min, 5 min, and 30 min are listed in Table 24.

Table 24: Peracetic acid (PAA) production in reactions containing triacetin and hydrogen peroxide in bicarbonate buffer (1 mM at pH 6.0 or 100 mM at pH 8.1) or in deionized water (pH 5.0) at 25 ⁰ C using heat-treated T. maritima wild- type, C277S or C277T variant perhydrolases.

Thermotoga triacetin H ₂ O ₂ NaHCO ₃ enzyme PAA, PAA ₁ PAA, maritima (mM) (mM) buffer concen. 1 min 5 min 30 min perhydrolase (mM) (μg/mL) (PPm) (ppm) (ppm) no enzyme 100 100 1.0 0 28 78 141 wild-type 100 100 1.0 75 434 494 608 wild -type 100 100 1.0 100 449 667 643

C277S 100 100 1.0 15 989 1554 1476

C277S 100 100 1.0 20 1301 2139 2131

C277T 100 100 1.0 15 1062 1513 1393

C277T 100 100 1.0 20 996 1430 1516

no enzyme 100 250 0 0 13 71 71 wild-type 100 250 0 75 512 535 533 wild-type 100 250 0 100 576 668 654

C277S 100 250 0 15 653 671 675

C277S 100 250 0 20 943 927 903

C277T 100 250 0 15 717 711 765

C277T 100 250 0 20 730 755 743

no enzyme 150 420 100 0 417 810 848 wiid-type 150 420 100 500 6303 8627 9237

C277S 150 420 100 100 7822 10349 10197